1-(pyrrlolidin-1-ylmethyl)-3-(pyrrol-2-ylmethylidene)-2-indolinone derivatives

ABSTRACT

The present invention is directed to 1-pyrrolidin-1-ylmethyl-3-(pyrrol-2-ylmethylidene)-2-indolinone derivatives that modulate the activity of protein kinases (“PKs”). Pharmaceutical compositions comprising these compounds, methods of treating diseases related to abnormal PK activity utilizing pharmaceutical compositions comprising these compounds and methods of preparing them are also disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority under 35 U.S.C. 119(e) to U.S.Provisional applications Serial No. 60/207,000 filed on May 24, 2000,and 60/225,045, filed on Aug. 11, 2000, the disclosures of which areincorporated herein by reference in their entirety.

BACKGROUND OF THE INVENTION

[0002] 1. Field of Invention

[0003] The present invention is directed to1-(pyrrolidin-1-ylmethyl)-3-(pyrrol-2-ylmethylidene)-2-indolinonederivatives that modulate the activity of protein kinases (“PKs”).Pharmaceutical compositions comprising these compounds, methods oftreating diseases related to abnormal PK activity utilizingpharmaceutical compositions comprising these compounds and methods ofpreparing them are also disclosed.

[0004] 2. State of the Art

[0005] Protein kinases (“PKs”) are enzymes that catalyze thephosphorylation of hydroxy groups on tyrosine, serine and threonineresidues of proteins. PKs can be conveniently broken down into twoclasses, the protein tyrosine kinases (PTKs) and the serine-threoninekinases (STKs). One of the prime aspects of PTK activity is theirinvolvement with growth factor receptors. Growth factor receptors arecell-surface proteins. When bound by a growth factor ligand, growthfactor receptors are converted to an active form which interacts withproteins on the inner surface of a cell membrane. This leads tophosphorylation on tyrosine residues of the receptor and other proteinsand to the formation inside the cell of complexes with a variety ofcytoplasmic signaling molecules that, in turn, effect numerous cellularresponses such as cell division (proliferation), cell differentiation,cell growth, expression of metabolic effects to the extracellularmicroenvironment, etc (See., Schlessinger and Ullrich (1992) Neuron9:303-391).

[0006] Growth factor receptors with PTK activity are known as receptortyrosine kinases (“RTKs”). They comprise a large family of transmembranereceptors with diverse biological activity. At present, at leastnineteen (19) distinct subfamilies of RTKs have been identified. Anexample of these is the subfamily designated the “HER” RTKs, whichinclude EGFR (epithelial growth factor receptor), HER2, HER3 and HER4.

[0007] Another RTK subfamily consists of insulin receptor (IR),insulin-like growth factor I receptor (IGF-1R) and insulin receptorrelated receptor (IRR). IR and IGF-1R interact with insulin, IGF-I andIGF-II to form a heterotetramer of two entirely extracellularglycosylated α subunits and two β subunits which cross the cell membraneand which contain the tyrosine kinase domain.

[0008] A third RTK subfamily is referred to as the platelet derivedgrowth factor receptor (“PDGFR”) group, which includes PDGFRα, PDGFRβ,CSFIR, c-kit and c-fms. Another group is the fetus liver kinase (“flk”)receptor subfamily. This group is believed to be made of up of kinaseinsert domain-receptor fetal liver kinase-1 (KDR/FLK-1), flk-1R, flk-4and fins-like tyrosine kinase 1 (flt-1).

[0009] A further member of the tyrosine kinase growth factor receptorfamily is the fibroblast growth factor (“FGF”) receptor subgroup. Thisgroup consists of four receptors, FGFR1-4, and seven ligands, FGF1-7.While not yet well defined, it appears that the receptors consist of aglycosylated extracellular domain containing a variable number ofimmunoglobin-like loops and an intracellular domain in which thetyrosine kinase sequence is interrupted by regions of unrelated aminoacid sequences.

[0010] Still another member of the tyrosine kinase growth factorreceptor family is the vascular endothelial growth factor (“VEGF”)receptor subgroup. VEGF is a dimeric glycoprotein similar to PDGF buthas different biological functions and target cell specificity in vivo.In particular, VEGF is presently thought to play an essential role isvasculogenesis and angiogenesis.

[0011] A more complete listing of the known RTK subfamilies is describedin Plowman et al., DN&P, 7(6):334-339 (1994) which is incorporated byreference, including any drawings, as if fully set forth herein.

[0012] In addition to the RTKs, there also exists a family of entirelyintracellular PTKs called “non-receptor tyrosine kinases” or “cellulartyrosine kinases.” This latter designation, abbreviated “CTK,” will beused herein. CTKs do not contain extracellular and transmembranedomains. At present, over 24 CTKs in 11 subfamilies (Src, Frk, Btk, Csk,Abl , Zap70, Fes, Fps, Fak, Jak and Ack) have been identified. The Srcsubfamily appear so far to be the largest group of CTKs and includesSrc, Yes, Fyn, Lyn, Lck, Blk, Hck, Fgr and Yrk. For a more detaileddiscussion of CTKs, see Bolen, Oncogene, 8:2025-2031 (1993), which isincorporated by reference, including any drawings, as if fully set forthherein.

[0013] The serine/threonine kinases, STKs, like the CTKs, arepredominantly intracellular although there are a few receptor kinases ofthe STK type. STKs are the most common of the cytosolic kinases; i.e.,kinases that perform their function in that part of the cytoplasm otherthan the cytoplasmic organelles and cytoskelton. The cytosol is theregion within the cell where much of the cell's intermediary metabolicand biosynthetic activity occurs; e.g., it is in the cytosol thatproteins are synthesized on ribosomes.

[0014] RTKs, CTKs and STKs have all been implicated in a host ofpathogenic conditions including, significantly, cancer. Other pathogenicconditions which have been associated with PTKs include, withoutlimitation, psoriasis, hepatic cirrhosis, diabetes, angiogenesis,restenosis, ocular diseases, rheumatoid arthritis and other inflammatorydisorders, immunological disorders such as autoimmune disease,cardiovascular disease such as atherosclerosis and a variety of renaldisorders.

[0015] With regard to cancer, two of the major hypotheses advanced toexplain the excessive cellular proliferation that drives tumordevelopment relate to functions known to be PK regulated. That is, ithas been suggested that malignant cell growth results from a breakdownin the mechanisms that control cell division and/or differentiation. Ithas been shown that the protein products of a number of proto-oncogenesare involved in the signal transduction pathways that regulate cellgrowth and differentiation. These protein products of proto-oncogenesinclude the extracellular growth factors, transmembrane growth factorPTK receptors (RTKs), cytoplasmic PTKs (CTKs) and cytosolic STKs,discussed above.

[0016] In view of the apparent link between PK-related cellularactivities and wide variety of human disorders, it is no surprise that agreat deal of effort is being expended in an attempt to identify ways tomodulate PK activity. For example, attempts have been made to identifysmall molecules which act as PK inhibitors. For example, bis-monocylic,bicyclic and heterocyclic aryl compounds (PCT WO 92/20642),vinylene-azaindole derivatives (PCT WO 94/14808) and1-cyclopropyl-4-pyridylquinolones (U.S. Pat. No. 5,330,992) have beendescribed as tyrosine kinase inhibitors. Styryl compounds (U.S. Pat. No.5,217,999), styryl-substituted pyridyl compounds (U.S. Pat. No.5,302,606), quinazoline derivatives (EP Application No. 0 566 266 A1),selenaindoles and selenides (PCT WO 94/03427), tricyclic polyhydroxyliccompounds (PCT WO 92/21660), and benzylphosphonic acid compounds (PCT WO91/15495). Additionally, a family of novel pyrrole-substituted2-indolinone compounds have been discovered which exhibit PK modulatingability and have a salutary effect against disorders related to abnormalPK activity (U.S. Pat. No. 5,792,783 and PCT Application Publication No.WO 99/61422). Administration of various species of pyrrole-substituted2-indolinone compounds has been shown to be an effective therapeuticapproach to cure many kinds of solid tumors. For example,3-(3,5-dimethyl-1H-pyrrol-2-ylmethylidene)-1,3-dihydro-indol-2-one, ahighly active selective inhibitor of the vascular endothelial growthfactor receptor (Flk-1/KDR), inhibits tyrosine kinase catalysis, tumorvascularization, and growth of multiple tumor types (Fong et al. (1999)Cancer Res. 59:99-106). These compounds, however, have highlipophilicity and low solubility in water and most common vehicles atphysiological pH limit their administration.

[0017] Accordingly, there is a need for PK inhibitors that do notexhibit such drawbacks. The present invention fulfills this and relatedneeds.

SUMMARY OF THE INVENTION

[0018] In one aspect, this invention is directed to a compound ofFormula (I):

[0019] wherein:

[0020] R³, R⁴, R⁵ and R⁶ are independently selected from the groupconsisting of hydrogen, alkyl, trihaloalkyl, cycloalkyl, alkenyl,alkynyl, aryl, heteroaryl, heteroalicyclic, hydroxy, alkoxy, aryloxy,mercapto, alkylthio, arylthio, sulfinyl, sulfonyl, S-sulfonamido,N-sulfonamido, trihalomethane-sulfonamido, carbonyl, C-carboxy,O-carboxy, C-amido, N-amido, cyano, nitro, halo, O-carbamyl, N-carbamyl,O-thiocarbamyl, N-thiocarbamyl, amino and —NR¹¹R¹² where R¹¹ and R¹² areindependently selected from the group consisting of hydrogen, alkyl,cycloalkyl, aryl, carbonyl, acetyl, sulfonyl, andtrifluoromethanesulfonyl, or R¹¹ and R¹², together with the nitrogenatom to which they are attached, combine to form a five- or six-memberheteroalicyclic ring provided that at least two of R³, R⁴, R⁵ and R⁶ arehydrogen; or

[0021] R³ and R⁴, R⁴ and R⁵, or R⁵ and R⁶ combine to form a six-memberaryl ring, a methylenedioxy or an ethylenedioxy group;

[0022] R⁷ is selected from the group consisting of hydrogen, alkyl,cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, heteroalicyclic,hydroxy, alkoxy, aryloxy, carbonyl, acetyl, C-amido, C-thioamido,amidino, C-carboxy, O-carboxy, sulfonyl, and trihalomethane-sulfonyl;

[0023] R⁸, R⁹ and R¹⁰ are independently selected from the groupconsisting of hydrogen, alkyl, trihaloalkyl, cycloalkyl, alkenyl,alkynyl, aryl, heteroaryl, heteroalicyclic, hydroxy, alkoxy, aryloxy,mercapto, alkylthio, arylthio, sulfinyl, sulfonyl, S-sulfonamido,N-sulfonamido, carbonyl, C-carboxy, O-carboxy, cyano, nitro, halo,O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-amido,N-amido, amino and —NR¹¹R¹², wherein R¹¹ and R¹² are as defined above;or a pharmaceutically acceptable salt thereof.

[0024] Preferably, R³, R⁵ and R⁶ are independently selected from thegroup consisting of hydrogen, alkyl, trihaloalkyl, cycloalkyl, alkenyl,alkynyl, aryl, heteroaryl, heteroalicyclic, hydroxy, alkoxy, aryloxy,mercapto, alkylthio, arylthio, sulfinyl, sulfonyl, S-sulfonamido,N-sulfonamido, trihalomethane-sulfonamido, carbonyl, C-carboxy,O-carboxy, C-amido, N-amido, cyano, nitro, halo, O-carbamyl, N-carbamyl,O-thiocarbamyl, N-thiocarbamyl, amino and —NR¹¹R¹² where R¹¹ and R¹² areindependently selected from the group consisting of hydrogen, alkyl,cycloalkyl, aryl, carbonyl, acetyl, sulfonyl, trifluoromethanesulfonyland, together with the nitrogen to which they are attached form, a five-or six-member heteroalicyclic ring; especially R³, R⁵, and R⁶ arehydrogen;

[0025] R⁷ is hydrogen;

[0026] R⁴ is hydrogen or halo, especially hydrogen, fluoro, or chloro,particularly hydrogen or fluoro;

[0027] R⁸ and R¹⁰ are independently unsubstituted lower alkyl,especially methyl; and

[0028] R⁹ is hydrogen, lower alkyl substituted with C-carboxy or—C(═O)NHR¹² wherein R¹² is lower alkyl substituted with amino orheteroalicyclic and optionally substituted with hydroxy; R⁹ ispreferably hydrogen, 3-carboxypropyl,(2-diethylaminoethyl)-aminocarbonyl, (2-ethylaminoethyl)aminocarbonyl,3-(morpholin-4-yl)propyl-aminocarbonyl,3-(morpholin-4-yl)-2-hydroxypropylaminocarbonyl; R⁹ is most preferablyhydrogen, 3-carboxypropyl, (2-diethylaminoethyl)aminocarbonyl, or(2-ethylaminoethyl)-aminocarbonyl.

[0029] A number of different preferences have been given above, andfollowing any one of these preferences results in a compound of thisinvention that is more presently preferred than a compound in which thatparticular preference is not followed. However, these preferences aregenerally independent [although some (alternative) preferences aremutually exclusive], and additive; and following more than one of thesepreferences may result in a more presently preferred compound than onein which fewer of the preferences are followed.

[0030] Presently preferred classes of compounds of this inventioninclude those where:

[0031] (a) R³, R⁴, R⁵, R⁶, R⁷, and R⁹ are hydrogen and R⁸ and R¹⁰ areunsubstituted lower alkyl, especially methyl.

[0032] (b) R³, R⁴, R⁵, R⁶, and R⁷ are hydrogen; R⁸ and R¹⁰ areunsubstituted lower alkyl, especially methyl; and R⁹ is lower alkylsubstituted with C-carboxy, especially 3-carboxypropyl.

[0033] Presently preferred compounds of this invention include:

[0034](3Z)-3-[(3,5-dimethyl-1H-pyrrol-2-yl)-methylidene]-1-(1-pyrrolidinylmethyl)-1,3-dihydro-2H-indol-2-one;and

[0035](3Z)-3-{[3,5-dimethyl-4-(3-carboxypropyl)-1H-pyrrol-2-yl]-methylidene}-1-(1-pyrrolidinylmethyl)-1,3-dihydro-2H-indol-2-one.

[0036] The compounds of the present invention convert in vivo tocompounds of Formula (II):

[0037] that exhibit PK modulating ability, in particular PK inhibitingability, and are therefore useful in treating disorders related toabnormal PK activity. The active compounds (II) formed from thecompounds of the present invention are described in U.S. Pat. No.5,792,783, PCT Application Publication No. WO 99/61422, and U.S. patentapplication Ser. No. 09/783,264, filed on Feb. 15, 2001, and titled“PYRROLE SUBSTITUTED 2-INDOLINONE AS PROTEIN KINASE INHIBITORS”, thedisclosures of which are hereby incorporated by reference.

[0038] The prodrug compounds of the present invention have advantagesover compounds of Formula (II) by virtue of improved aqueous solubilityand formulability. For example, Applicants have discovered that theN-pyrrolidin-1-ylmethyl prodrug of compound (II) where R³-R⁷ and R⁹ arehydrogen and R⁸ and R¹⁰ are methyl provides unexpected increased aqueoussolubility over the parent compound thus making it particularly suitablefor intravenous (IV) formulations. It is contemplated that similarenhanced solubility will be observed for other compounds of Formula (I)carrying the pyrrolidin-1-ylmethyl moiety at the nitrogen of theindolinone ring. A general description of the advantages and uses ofprodrugs as pharmaceutically useful compounds is given in an article byWaller and George in Br. J Clin. Pharmac., Vol. 28, pp. 497-507, 1989.

[0039] In a second aspect this invention is directed to a pharmaceuticalcomposition comprising one or more compound(s) of Formula (I) or apharmaceutically acceptable salt thereof and a pharmaceuticallyacceptable excipient.

[0040] In a third aspect, this invention is directed to a method oftreating diseases mediated by abnormal protein kinase activity, inparticular, receptor tyrosine kinases (RTKs), non-receptor proteintyrosine kinases (CTKs) and serine/threonine protein kinases (STKs), inan organism, in particular humans, which method comprises administeringto said organism a pharmaceutical composition comprising a compound ofFormula (I) or a pharmaceutically acceptable salt thereof and apharmaceutically acceptable excipient. Such diseases include by way ofexample and not limitation, cancer, diabetes, hepatic cirrhosis,cardiovascular disease such as atherosclerosis, angiogenesis,immunological disease such as autoimmune disease (e.g., AIDS and lupus)and renal disease. Specifically, the diseases mediated by EGF, HER2,HER3, HER4, IR, IGF-1R, IRR, PDGFRα, PDGFRβ, CSFIR, C-Kit, C-fins,Flk-1R, Flk4, KDR/Flk-1, Flt-1, FGFR-1R, FGFR-2R, FGFR-3R, FGFR-4R, Src,Frk, Btk, Csk, Abl , ZAP70, Fes/Fps, Fak, Jak, Ack, Yes, Fyn, Lyn, Lck,Blk, Hck, Fgr, Yrk, CDK2 and Raf.

[0041] In a fourth aspect, this invention is directed to a method ofmodulating the catalytic activity (e.g., inhibiting the catalyticactivity) of PKs, in particular receptor tyrosine kinases (RTKs),non-receptor protein tyrosine kinases (CTKs) and serine/threonineprotein kinases (STKs), using a compound of this invention or apharmaceutical composition comprising a compound of this invention and apharmaceutically acceptable excipient. The method may be carried out invitro or in vivo. In particular, the receptor protein kinase whosecatalytic activity is modulated by a compound of this invention isselected from the group consisting of EGF, HER2, HER3, HER4, IR, IGF-1R,IRR, PDGFRα, PDGFRβ, CSFIR, C-Kit, C-fms, Flk-1R, Flk4, KDR/Flk-1,Flt-1, FGFR-1R, FGFR-2R, FGFR-3R and FGFR-4R. The cellular tyrosinekinase whose catalytic activity is modulated by a compound of thisinvention is selected from the group consisting of Src, Frk, Btk, Csk,Abl, ZAP70, Fes/Fps, Fak, Jak, Ack, Yes, Fyn, Lyn, Lck, Blk, Hck, Fgrand Yrk. The serine-threonine protein kinase whose catalytic activity ismodulated by a compound of this invention is selected from the groupconsisting of CDK2 and Raf.

[0042] In a fifth aspect, this invention is directed to the use of acompound of Formula (I) in the preparation of a medicament useful in thetreatment of a disease mediated by abnormal PK activity.

[0043] In a sixth aspect, this invention is directed to a method ofpreparing a compound of Formula (I) which method comprises reacting acompound of Formula (II)

[0044] with an pyrrolidine in the presence of formaldehyde;

[0045] optionally modifying any of the R³-R¹⁰ groups; and

[0046] optionally preparing an acid addition salt thereof.

DETAILED DESCRIPTION OF THE INVENTION Definitions

[0047] Unless otherwise stated the following terms used in thespecification and claims have the meanings discussed below:

[0048] “Alkyl” refers to a saturated aliphatic hydrocarbon includingstraight chain, or branched chain groups. Preferably, the alkyl grouphas 1 to 20 carbon atoms (whenever a numerical range; e.g., “1-20”, isstated herein, it means that the group, in this case the alkyl group,may contain 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc. up toand including 20 carbon atoms). More preferably, it is a medium sizealkyl having 1 to 10 carbon atoms. Most preferably, it is a lower alkylhaving 1 to 4 carbon atoms e.g., methyl, ethyl, n-propyl, isopropyl,butyl, iso-butyl, tert-butyl and the like. The alkyl group may besubstituted or unsubstituted. When substituted, the substituent group(s)is preferably one or more, more preferably one or two groups,individually selected from the group consisting of cycloalkyl, aryl,heteroaryl, heteroalicyclic, hydroxy, alkoxy, aryloxy, mercapto,alkylthio, arylthio, cyano, halo, carbonyl, thiocarbonyl, O-carbamyl,N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-amido, N-amido, C-carboxy,O-carboxy, nitro, silyl, amino, ammonium and —NR¹³R¹⁴ where R¹³ and R¹⁴are independently selected from the group consisting of hydrogen,unsubstituted alkyl, alkyl, cycloalkyl, aryl, carbonyl, acetyl,sulfonyl, amino, and trifluoromethanesulfonyl, or R¹³ and R¹⁴, togetherwith the nitrogen atom to which they are attached, combine to form afive- or six-member heteroalicyclic ring. More preferably, thesubstituent is hydroxy, amino, or —NR¹³R¹⁴ where R¹³ and R¹⁴ areindependently selected from the group consisting of unsubstituted loweralkyl, lower alkyl substituted with amino or hydroxy, or R¹³ and R¹⁴,together with the nitrogen atom to which they are attached, combine toform pyrrolidine, morpholine, or piperazine.

[0049] A “cycloalkyl” group refers to an all-carbon monocyclic ring(i.e., rings which share an adjacent pair of carbon atoms) of 3 to 6ring atoms wherein one of more of the rings does not have a completelyconjugated pi-electron system e.g., cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl, cyclobutenyl, cyclopentenyl, cyclohexenyl, andthe like. Examples, without limitation, of cycloalkyl groups arecyclopropane, cyclobutane, cyclopentane, cyclopentene, cyclohexane,adamantane, cyclohexadiene, cycloheptane and, cycloheptatriene. Acycloalkyl group may be substituted or unsubstituted. When substituted,the substituent group(s) is preferably one or more, more preferably oneor two groups, individually selected from unsubstituted alkyl, alkyl,aryl, heteroaryl, unsubstituted heteroalicyclic, heteroalicyclic,hydroxy, alkoxy, aryloxy, mercapto, alkylthio, arylthio, cyano, halo,carbonyl, thiocarbonyl, C-carboxy, O-carboxy, O-carbamyl, N-carbamyl,C-amido, N-amido, nitro, amino and —NR¹³R¹⁴, with R¹³ and R¹⁴ as definedabove.

[0050] An “alkenyl” group refers to an alkyl group, as defined herein,consisting of at least two carbon atoms and at least one carbon-carbondouble bond e.g., ethenyl, propenyl, butenyl or pentenyl and theirstructural isomeric forms such as 1- or 2-propenyl, 1-, 2-, or 3-butenyland the like.

[0051] An “alkynyl” group refers to an alkyl group, as defined herein,consisting of at least two carbon atoms and at least one carbon-carbontriple bond e.g., acetylene, ethnyl, propynyl, butynyl, or pentnyl andtheir structural isomeric forms as described above.

[0052] An “aryl” group refers to an all-carbon monocyclic or fused-ringpolycyclic (i.e., rings which share adjacent pairs of carbon atoms)groups of 6 to 12 ring atoms and having a completely conjugatedpi-electron system. Examples, without limitation, of aryl groups arephenyl, naphthalenyl and anthracenyl. The aryl group may be substitutedor unsubstituted. When substituted, the substituted group(s) ispreferably one or more, more preferably one, two, or three substituents,independently selected from the group consisting of halo, trihalomethyl,alkyl, hydroxy, alkoxy, aryloxy, mercapto, alkylthio, arylthio, cyano,nitro, carbonyl, thiocarbonyl, C-carboxy, O-carboxy, O-carbamyl,N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-amido, N-amido, sulfinyl,sulfonyl, amino and —NR¹³R¹⁴, with R¹³ and R¹⁴ as defined above.Preferably the substituent(s) is/are independently selected from chloro,fluoro, bromo, methyl, ethyl, propyl including all its isomeric forms,butyl including all its isomeric forms, hydroxy, methoxy, phenoxy, thio,methylthio, phenylthio, cyano, nitro, carboxy, methoxycarbonyl, oramino.

[0053] A “heteroaryl” group refers to a monocyclic or fused aromaticring (i.e., rings which share an adjacent pair of atoms) of 5 to 9 ringatoms in which one, two, three or four ring atoms are selected from thegroup consisting of nitrogen, oxygen and sulfur and the rest beingcarbon. Examples, without limitation, of heteroaryl groups are pyrrole,furan, thiophene, imidazole, oxazole, thiazole, pyrazole, tetrazole,pyridine, pyrimidine, quinoline, isoquinoline, purine and carbazole. Theheteroaryl group may be substituted or unsubstituted. When substituted,the substituted group(s) is preferably one or more, more preferably oneor two substituents, independently selected from the group consisting ofalkyl, cycloalkyl, halo, trihalomethyl, hydroxy, alkoxy, aryloxy,mercapto, alkylthio, arylthio, cyano, nitro, carbonyl, thiocarbonyl,sulfonamido, C-carboxy, O-carboxy, sulfinyl, sulfonyl, O-carbamyl,N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-amido, N-amido, amino and—NR¹³R¹⁴, with R¹³ and R¹⁴ as defined above. Preferably thesubstituent(s) is/are independently selected from chloro, fluoro, bromo,methyl, ethyl, propyl including all its isomeric forms, butyl includingall its isomeric forms, hydroxy, methoxy, phenoxy, thio, methylthio,phenylthio, cyano, nitro, carboxy, methoxycarbonyl, or ammo.

[0054] A “heteroalicyclic” group refers to a monocyclic or fused ring of4 to 9 ring atoms containing one, two, or three heteroatoms in the ringwhich are selected from the group consisting of nitrogen, oxygen and—S(O)_(n) where n is 0-2, the remaining ring atoms being carbon. Therings may also have one or more double bonds. However, the rings do nothave a completely conjugated pi-electron system. Examples, withoutlimitation, of heteroalicyclic groups are pyrrolidine, piperidine,piperazine, morpholine, imidazolidine, tetrahydropyridazine,tetrahydrofuran, thiomorpholine, tetrahydropyridine, and the like. Theheteroalicyclic ring may be substituted or unsubstituted. Whensubstituted, the substituted group(s) is preferably one or more, morepreferably one, two, or three substituents, independently selected fromthe group consisting of alkyl, cycloalkyl, halo, trihalomethyl, hydroxy,alkoxy, aryloxy, mercapto, alkylthio, arylthio, cyano, nitro, carbonyl,thiocarbonyl, C-carboxy, O-carboxy, O-carbamyl, N-carbamyl,O-thiocarbamyl, N-thiocarbamyl, sulfinyl, sulfonyl, C-amido, N-amido,amino and —NR¹³R¹⁴, with R¹³ and R¹⁴ as defined above. Preferably thesubstituent(s) is/are independently selected from chloro, fluoro, bromo,methyl, ethyl, propyl including all its isomeric forms, butyl includingall its isomeric forms, hydroxy, methoxy, phenoxy, thio, methylthio,phenylthio, cyano, nitro, carboxy, methoxycarbonyl, or amino.

[0055] A “hydroxy” group refers to an —OH group.

[0056] An “alkoxy” group refers to an —O-unsubstituted alkyl,—O-substituted alkyl and an —O-unsubstitutedcycloalkyl group, as definedherein. Examples include and are not limited to methoxy, ethoxy,propoxy, butoxy, cyclopropyloxy, and the like, preferably methoxy.

[0057] An “aryloxy” group refers to both an —O-aryl and an —O-heteroarylgroup, as defined herein. Examples include and are not limited tophenoxy, napthyloxy, pyridyloxy, furanyloxy, and the like.

[0058] A “mercapto” group refers to an —SH group.

[0059] A “alkylthio” group refers to both an S-alkyl and an—S-cycloalkyl group, as defined herein. Examples include and are notlimited to methylthio, ethylthio, and the like.

[0060] A “arylthio” group refers to both an —S-aryl and an —S-heteroarylgroup, as defined herein. Examples include and are not limited tophenylthio, napthylthio, pyridylthio, furanylthio, and the like.

[0061] A “sulfinyl” group refers to a 13 S(═O)—R″ group wherein, inaddition to being as defined below, R″ may also be a hydroxy group,e.g., methylsulfinyl, phenylsulfinyl, and the like.

[0062] A “sulfonyl” group refers to a —S(═O)₂R″ group wherein, inaddition to being as defined below, R″ may also be a hydroxy group e.g.,methylsulfonyl, ethylsulfonyl, phenylsulfonyl, and the like.

[0063] A “trihalomethyl” group refers to a —CX₃ group wherein X is ahalo group as defined herein e.g., trifluoromethyl, trichloromethyl,tribromomethyl, dichlorofluoromethyl, and the like.

[0064] A “trihalomethanesulfonyl” group refers to a X₃CS(═O)₂— groupswith X as defined above, e.g., trifluoromethylsulfonyl,trichloromethylsulfonyl, tribromomethylsulfonyl, and the like.

[0065] A “trihalomethanesulfonylamido” group refers to a —NH—S(═O)₂Rgroups wherein R is trihalomethyl as defined above. “Carbonyl” and“acyl” are used interchangeably herein to refer to a —C(═O)—R″ group,where R″ is selected from the group consisting of hydrogen, alkyl,cycloalkyl, aryl, heteroaryl (bonded through a ring carbon) andheteroalicyclic (bonded through a ring carbon), as defined herein.Representative examples include and are not limited to acetyl,propionyl, benzoyl, formyl, cyclopropylcarbonyl, pyridinylcarbonyl,pyrrolidin-1-ylcarbonyl, and the like

[0066] An “aldehyde” group refers to a carbonyl group where R″ ishydrogen.

[0067] A “thiocarbonyl” group refers to a —C(═S)—R″ group, with R″ asdefined herein.

[0068] A “C-carboxy” group refers to a —C(═O)O—R″ group, with R″ asdefined herein e.g., —COOH, methoxycarbonyl, ethoxycarbonyl,benzyloxycarbonyl, and the like.

[0069] An “O-carboxy” group refers to a —OC(═O)R″ group, with R″ asdefined herein e.g., methylcarbonyloxy, phenylcarbonyloxy,benzylcarbonyloxy, and the like.

[0070] An “ester” group refers to a —C(═O)O—R″ group with R″ as definedherein except that R″ cannot be hydrogen e.g., methoxycarbonyl,benzyloxycarbonyl, and the like.

[0071] An “acetyl” group refers to a —C(═O)CH₃ group.

[0072] A “carboxylic acid” group refers to a C-carboxy group in which R″is hydrogen.

[0073] A “halo” group refers to fluorine, chlorine, bromine or iodine.

[0074] A “cyano” group refers to a —C≡N group.

[0075] A “nitro” group refers to a —NO₂ group.

[0076] A “methylenedioxy” group refers to —OCH₂O— group where the twooxygen atoms are bonded to adjacent carbon atoms.

[0077] An “ethylenedioxy” group refers to —OCH₂CH₂O— where the twooxygen atoms are bonded to adjacent carbon atoms.

[0078] An “S-sulfonamido” group refers to a —S(═O)₂NR¹³R¹⁴ group, withR¹³ and R¹⁴ as defined herein. Representative examples include and arenot limited to dimethylaminosulfonyl, aminosulfonyl,phenylmethylaminosulfonyl, phenylaminosulfonyl, and the like.

[0079] An “N-sulfonamido” group refers to a —NR¹³S(═O)₂R¹⁴ group, withR¹³ and R¹⁴ as defined herein e.g., methylsulfonylamino,ethylsulfonylamino, phenylsulfonylamino, benzylsulfonylamino, and thelike.

[0080] An “O-carbamyl” group refers to a —OC(═O)NR¹³R¹⁴ group with R¹³and R¹⁴ as defined herein.

[0081] An “N-carbamyl” group refers to a R¹⁴OC(═O)NR¹³— group, with R¹³and R¹⁴ as defined herein.

[0082] An “O-thiocarbamyl” group refers to a —OC(═S)NR¹³R¹⁴ group withR¹³ and R¹⁴ as defined herein.

[0083] An “N-thiocarbamyl” group refers to a R¹⁴OC(═S)NR¹³— group, withR¹³ and R¹⁴ as defined herein.

[0084] An “amino” group refers to an —NR¹³R¹⁴ group, wherein R¹³ and R¹⁴are independently hydrogen or unsubstituted lower alkyl e.g., —NH₂,dimethylamino, diethylamino, ethylamino, methylamino, and the like.

[0085] A “C-amido” group refers to a —C(═O)NR¹³R¹⁴ group with R¹³ andR¹⁴ as defined herein. Preferably R¹³ is hydrogen or unsubstituted loweralkyl and R¹⁴ is hydrogen, lower alkyl optionally substituted withheteroalicyclic, hydroxy, or amino. For example, —C (═O)NR¹³R¹⁴ may beaminocarbonyl, dimethylaminocarbonyl, diethylaminocarbonyl,diethylaminoethylaminocarbonyl, ethylaminoethylaminocarbonyl,2-morpholinoethylaminocarbonyl, 3-morpholinopropylaminocarbonyl,3-morpholino-2-hydroxypropylaminocarbonyl, and the like.

[0086] An “N-amido” group refers to a R¹⁴C(═O)NR¹³— group, with R¹³ andR¹⁴ as defined herein e.g., acetylamino, and the like.

[0087] A “ammonium” group refers to a —⁺NR¹⁵R¹⁶R¹⁷ group, wherein R¹⁵and R¹⁶ are independently selected from the group consisting of alkyl,cycloalkyl, aryl, and heteroaryl, and R¹⁷ is selected from the groupconsisting of hydrogen, alkyl, cycloalkyl, aryl, and heteroaryl.

[0088] A “amidino” group refers to a R¹⁵R¹⁶NC(═NR¹⁷)— group, with R¹⁵,R¹⁶ and R¹⁷ as defined herein.

[0089] A “morpholino” group refers to a group having the chemicalstructure

[0090] A “piperazinyl” group refers to a group having the chemicalstructure:

[0091] The terms “indolinone”, “2-indolinone” and “indolin-2-one” areused interchangeably herein to refer to a molecule having the chemicalstructure:

[0092] “Pyrrole” refers to a molecule having the chemical structure:

[0093] “Pyrrole-substituted 2-indolinone” and“3-pyrrol-1-yl-2-indolinone” are used interchangeably herein to refer toa chemical compound having the general structure shown in Formula II.

[0094] A “prodrug” refers to an agent which is converted into the parentdrug in vivo. Prodrugs are often useful because, in some situations,they may be easier to administer than the parent drug. They may, forinstance, be bioavailable by oral administration whereas the parent drugis not. The prodrug may also have improved solubility in pharmaceuticalcompositions over the parent drug. A prodrug may be converted into theparent drug by various mechanisms, including enzymatic processes andmetabolic hydrolysis. See Harper, “Drug Latentiation” in Jucker, ed.Progress in Drug Research 4:221-294 (1962); Morozowich et al.,“Application of Physical Organic Principles to Prodrug Design” in E. B.Roche ed. Design of Biopharmaceutical Properties through Prodrugs andAnalogs, APHA Acad. Pharm. Sci. (1977); Bioreversible Carriers in Drugin Drug Design, Theory and Application, E. B. Roche, ed., APHA Acad.Pharm. Sci. (1987); Design of Prodrugs, H. Bundgaard, Elsevier (1985);Wang et al. “Prodrug approaches to the improved delivery of peptidedrug” in Curr. Pharm. Design. 5(4):265-287 (1999); Pauletti et al.(1997) Improvement in peptide bioavailability: Peptidomimetics andProdrug Strategies, Adv. Drug. Delivery Rev. 27:235-256; Mizen et al.(1998) “The Use of Esters as Prodrugs for Oral Delivery of β-Lactamantibiotics,” Pharm. Biotech. 11,:345-365; Gaignault et al. (1996)“Designing Prodrugs and Bioprecursors I. Carrier Prodrugs,” Pract. Med.Chem. 671-696; Asgharnejad, “Improving Oral Drug Transport”, inTransport Processes in Pharmaceutical Systems, G. L. Amidon, P. I. Leeand E. M. Topp, Eds., Marcell Dekker, p. 185-218 (2000); Balant et al.,“Prodrugs for the improvement of drug absorption via different routes ofadministration”, Eur. J Drug Metab. Pharmacokinet., 15(2): 143-53(1990); Balimane and Sinko, “Involvement of multiple transporters in theoral absorption of nucleoside analogues”, Adv. Drug Delivery Rev.,39(1-3): 183-209 (1999); Browne, “Fosphenytoin (Cerebyx)”, Clin.Neuropharmacol. 20(1): 1-12 (1997); Bundgaard, “Bioreversiblederivatization of drugs—principle and applicability to improve thetherapeutic effects of drugs”, Arch. Pharm. Chemi 86(I): 1-39 (1979);Bundgaard H. “Improved drug delivery by the prodrug approach”,Controlled Drug Delivery 17: 179-96 (1987); Bundgaard H. “Prodrugs as ameans to improve the delivery of peptide drugs”, Adv. Drug Delivery Rev.8(1): 1-38 (1992); Fleisher et al. “Improved oral drug delivery:solubility limitations overcome by the use of prodrugs”, Adv. DrugDelivery Rev. 19(2): 115-130 (1996); Fleisher et al. “Design of prodrugsfor improved gastrointestinal absorption by intestinal enzymetargeting”, Methods Enzymol. 112 (Drug Enzyme Targeting, Pt. A): 360-81,(1985); Farquhar D, et al., “Biologically ReversiblePhosphate-Protective Groups”, J. Pharm. Sci., 72(3): 324-325 (1983);Freeman S, et al., “Bioreversible Protection for the Phospho Group:Chemical Stability and Bioactivation of Di(4-acetoxy-benzyl)Methylphosphonate with Carboxyesterase,” J. Chem. Soc., Chem. Commun.,875-877 (1991); Friis and Bundgaard, “Prodrugs of phosphates andphosphonates: Novel lipophilic alpha-acyloxyalkyl ester derivatives ofphosphate- or phosphonate containing drugs masking the negative chargesof these groups”, Eur. J. Pharm. Sci. 4: 49-59 (1996); Gangwar et al.,“Pro-drug, molecular structure and percutaneous delivery”, Des.Biopharm. Prop. Prodrugs Analogs, [Symp.] Meeting Date 1976, 409-21.(1977); Nathwani and Wood, “Penicillins: a current review of theirclinical pharmacology and therapeutic use”, Drugs 45(6): 866-94 (1993);Sinhababu and Thakker, “Prodrugs of anticancer agents”, Adv. DrugDelivery Rev. 19(2): 241-273 (1996); Stella et al., “Prodrugs. Do theyhave advantages in clinical practice?”, Drugs 29(5): 455-73 (1985); Tanet al. “Development and optimization of anti-HIV nucleoside analogs andprodrugs: A review of their cellular pharmacology, structure-activityrelationships and pharmacokinetics”, Adv. Drug Delivery Rev. 39(1-3):117-151 (1999); Taylor, “Improved passive oral drug delivery viaprodrugs”, Adv. Drug Delivery Rev., 19(2): 131-148 (1996); Valentino andBorchardt, “Prodrug strategies to enhance the intestinal absorption ofpeptides”, Drug Discovery Today 2(4): 148-155 (1997); Wiebe and Knaus,“Concepts for the design of anti-HIV nucleoside prodrugs for treatingcephalic HIV infection”, Adv. Drug Delivery Rev.: 39(1-3):63-80 (1999);Waller et al., “Prodrugs”, Br. J Clin. Pharmac. 28: 497-507 (1989).

[0095] The compounds of this invention may possess one or more chiralcenters, and can therefore be produced as individual stereoisomers or asmixtures of stereoisomers, depending on whether individual stereoisomersor mixtures of stereoisomers of the starting materials are used. Unlessindicated otherwise, the description or naming of a compound or group ofcompounds is intended to include both the individual stereoisomers ormixtures (racemic or otherwise) of stereoisomers. Methods for thedetermination of stereochemistry and the separation of stereoisomers arewell known to a person of ordinary skill in the art [see the discussionin Chapter 4 of March J: Advanced Organic Chemistry, 4th ed. John Wileyand Sons, New York, N.Y., 1992].

[0096] The chemical formulae referred to herein may exhibit thephenomena of tautomerism and structural isomerism. For example, thecompounds described herein may adopt an E or a Z configuration about thedouble bond connecting the 2-indolinone moiety to the pyrrole moiety orthey may be a mixture of E and Z. This invention encompasses anytautomeric or structural isomeric form and mixtures thereof.

[0097] The term “method” refers to manners, means, techniques andprocedures for accomplishing a given task including, but not limited to,those manners, means, techniques and procedures either known to, orreadily developed from known manners, means, techniques and proceduresby, practitioners of the chemical, pharmaceutical, biological,biochemical and medical arts.

[0098] As used herein, the term “modulation” or “modulating” refers tothe alteration of the catalytic activity of RTKs, CTKs and STKs. Inparticular, modulating refers to the activation of the catalyticactivity of RTKs, CTKs and STKs, preferably the activation or inhibitionof the catalytic activity of RTKs, CTKs and STKs, depending on theconcentration of the compound or salt to which the RTK, CTK or STK isexposed or, more preferably, the inhibition of the catalytic activity ofRTKs, CTKs and STKs.

[0099] The term “catalytic activity” as used herein refers to the rateof phosphorylation of tyrosine under the influence, direct or indirect,of RTKs and/or CTKs or the phosphorylation of serine and threonine underthe influence, direct or indirect, of STKs.

[0100] The term “contacting” as used herein refers to bringing acompound of this invention and a target PK together in such a mannerthat the compound can affect the catalytic activity of the PK, eitherdirectly, i.e., by interacting with the kinase itself, or indirectly,i.e., by interacting with another molecule on which the catalyticactivity of the kinase is dependent. Such “contacting” can beaccomplished in vitro, i.e., in a test tube, a petri dish or the like.In a test tube, contacting may involve only a compound and a PK ofinterest or it may involve whole cells. Cells may also be maintained orgrown in cell culture dishes and contacted with a compound in thatenvironment. In this context, the ability of a particular compound toaffect a PK related disorder, i.e., the IC₅₀ of the compound, definedbelow, can be determined before use of the compounds in vivo with morecomplex living organisms is attempted. For cells outside the organism,multiple methods exist, and are well-known to those skilled in the art,to get the PKs in contact with the compounds including, but not limitedto, direct cell microinjection and numerous transmembrane carriertechniques.

[0101] “In vitro” refers to procedures performed in an artificialenvironment such as, e.g., without limitation, in a test tube or culturemedium. The skilled artisan will understand that, for example, anisolated PK may be contacted with a modulator in an in vitroenvironment. Alternatively, an isolated cell may be contacted with amodulator in an in vitro environment.

[0102] As used herein, “in vivo” refers to procedures performed within aliving organism such as, without limitation, a mouse, rat, rabbit,ungulate, bovine, equine, porcine, canine, feline, primate, or human.

[0103] As used herein, “PK related disorder,” “PK driven disorder,” and“abnormal PK activity” all refer to a condition characterized byinappropriate, i.e., under or, more commonly, over PK catalyticactivity, where the particular PK can be an RTK, a CTK or an STK.Inappropriate catalytic activity can arise as the result of either: (1)PK expression in cells which normally do not express PKs, (2) increasedPK expression leading to unwanted cell proliferation, differentiationand/or growth, or, (3) decreased PK expression leading to unwantedreductions in cell proliferation, differentiation and/or growth.Over-activity of a PK refers to either amplification of the geneencoding a particular PK or production of a level of PK activity whichcan correlate with a cell proliferation, differentiation and/or growthdisorder (that is, as the level of the PK increases, the severity of oneor more of the symptoms of the cellular disorder increases).Under-activity is, of course, the converse, wherein the severity of oneor more symptoms of a cellular disorder increase as the level of the PKactivity decreases.

[0104] The term “organism” refers to any living entity comprised of atleast one cell. A living organism can be as simple as, for example, asingle eukaryotic cell or as complex as a mammal, including a humanbeing.

[0105] The term “therapeutically effective amount” as used herein refersto that amount of the compound being administered which will relieve tosome extent one or more of the symptoms of the disorder being treated.In reference to the treatment of cancer, a therapeutically effectiveamount refers to that amount which has the effect of (1) reducing thesize of the tumor, (2) inhibiting (that is, slowing to some extent,preferably stopping) tumor metastasis, (3) inhibiting to some extent(that is, slowing to some extent, preferably stopping) tumor growth,and/or, (4) relieving to some extent (or, preferably, eliminating) oneor more symptoms associated with the cancer.

[0106] “Pharmaceutically acceptable salt” refers to those salts whichretain the biological effectiveness and properties of the free bases andwhich are obtained by reaction with inorganic or organic acids such ashydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,phosphoric acid, methanesulfonic acid, ethanesulfonic acid,p-toluenesulfonic acid, salicylic acid, malic acid, citric acid, maleicacid, succinic acid, tartaric acid, and the like.

[0107] A “pharmaceutical composition” refers to a mixture of one or moreof the compounds described herein, or a pharmaceutically acceptablesalts thereof, with other chemical components, such as physiologicallyacceptable carriers and excipients. The purpose of a pharmaceuticalcomposition is to facilitate administration of a compound to anorganism.

[0108] As used herein, a “pharmaceutically acceptable carrier” refers toa carrier or diluent that does not cause significant irritation to anorganism and does not abrogate the biological activity and properties ofthe administered compound.

[0109] An “excipient” refers to an inert substance added to apharmaceutical composition to further facilitate administration of acompound. Examples, without limitation, of excipients include calciumcarbonate, calcium phosphate, various sugars and types of starch,cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.

[0110] “Treating” or “treatment” of a disease includes preventing thedisease from occurring in an animal that may be predisposed to thedisease but does not yet experience or exhibit symptoms of the disease(prophylactic treatment), inhibiting the disease (slowing or arrestingits development), providing relief from the symptoms or side-effects ofthe disease (including palliative treatment), and relieving the disease(causing regression of the disease). With regard to cancer, these termssimply mean that the life expectancy of an individual affected with acancer will be increased or that one or more of the symptoms of thedisease will be reduced.

[0111] By “monitoring” is meant observing or detecting the effect ofcontacting a compound with a cell expressing a particular PK. Theobserved or detected effect can be a change in cell phenotype, in thecatalytic activity of a PK or a change in the interaction of a PK with anatural binding partner. Techniques for observing or detecting sucheffects are well-known in the art. For example, the catalytic activityof a PK may be observed by determining the rate or amount ofphosphorylation of a target molecule. The above-referenced effect isselected from a change or an absence of change in a cell phenotype, achange or absence of change in the catalytic activity of said proteinkinase or a change or absence of change in the interaction of saidprotein kinase with a natural binding partner in a final aspect of thisinvention.

[0112] “Cell phenotype” refers to the outward appearance of a cell ortissue or the biological function of the cell or tissue. Examples,without limitation, of a cell phenotype are cell size, cell growth, cellproliferation, cell differentiation, cell survival, apoptosis, andnutrient uptake and use. Such phenotypic characteristics are measurableby techniques well-known in the art.

[0113] A “natural binding partner” refers to a polypeptide that binds toa particular PK in a cell. Natural binding partners can play a role inpropagating a signal in a PK-mediated signal transduction process. Achange in the interaction of the natural binding partner with the PK canmanifest itself as an increased or decreased concentration of thePK/natural binding partner complex and, as a result, in an observablechange in the ability of the PK to mediate signal transduction.

GENERAL SYNTHETIC SCHEME

[0114] The starting materials and reagents used in preparing thesecompounds are either available from commercial suppliers such as AldrichChemical Co., (Milwaukee, Wis.), Bachem (Torrance, Calif.), or Sigma(St. Louis, Mo.) or are prepared by methods known to those skilled inthe art following procedures set forth in references such as Fieser andFieser's Reagents for Organic Synthesis, Volumes 1-17 (John Wiley andSons, 1991); Rodd's Chemistry of Carbon Compounds, Volumes 1-5 andSupplementals (Elsevier Science Publishers, 1989); Organic Reactions,Volumes 1-40 (John Wiley and Sons, 1991), March's Advanced OrganicChemistry, (John Wiley and Sons, 4th Edition) and Larock's ComprehensiveOrganic Transformations (VCH Publishers Inc., 1989). These schemes aremerely illustrative of some methods by which the compounds of thisinvention can be synthesized, and various modifications to these schemescan be made and will be suggested to one skilled in the art havingreferred to this disclosure. The starting materials and theintermediates of the reaction may be isolated and purified if desiredusing conventional techniques, including but not limited to filtration,distillation, crystallization, chromatography and the like. Suchmaterials may be characterized using conventional means, includingphysical constants and spectral data. Unless specified to the contrary,the reactions described herein take place at atmospheric pressure over atemperature range from about −78° C. to about 150° C., more preferablyfrom about 0° C. to about 125° C. and most preferably at about room (orambient) temperature, e.g., about 20° C.

[0115] Compounds of Formula (I) may be prepared as illustrated anddescribed below:

[0116] A compound of Formula (I) where R³-R¹⁰ are as described in theSummary of the Invention can be prepared by reacting a compound offormula (II) with formaldehyde, acetaldehyde and pyrrolidine.

[0117] The solvent in which the reaction is carried out may be a proticor an aprotic solvent, preferably it is a protic solvent such as analcohol e.g., methanol or ethanol, or an aqueous alcohol. The reactionmay be carried out at temperatures greater than room temperature. Thetemperature is generally from about 20° C. to about 100° C., preferablyabout 40° C. to about 80° C. By “about” is meant that the temperaturerange is preferably within 10 degrees Celsius of the indicatedtemperature, more preferably within 5° C. of the indicated temperatureand, most preferably, within 2 degrees Celsius of the indicatedtemperature. Thus, for example, by “about 60° C.” is meant 60° C.±10°C., preferably 60° C.±5° C. and most preferably, 60° C.±2° C.

[0118] Compounds of Formula (II) can be prepared by methods well knownin the art. For example, compound (II) where R³-R⁶, R⁷, and R⁹ arehydrogen and R⁸ and R¹⁰ are methyl can be prepared by following theprocedure described in U.S. Pat. No. 5,792,783, at column 22, lines60-67, the disclosure of which is incorporated herein by reference.Other compounds of Formula (II) can be prepared as described in U.S.Pat. No. 5,792,783, PCT Application Publication No. WO 99/61422, andU.S. patent application Ser. No. 09/783,264, filed on Feb. 15, 2001, andtitled “PYRROLE SUBSTITUTED 2-INDOLINONE AS PROTEIN KINASE INHIBITORS”,the disclosures of which are hereby incorporated by reference.

[0119] The preparation of compounds of Formula (I) may further includethe step of removing a protecting group. “Protecting group” refers to agroup used to render a reactive moiety inert until removal of the group.Reactive moieties are well known to the skilled artisan; preferredreactive moieties include reactive nitrogen, oxygen, sulfur, carboxyland carbonyl groups. Exemplary nitrogen protecting groups include, butare not limited to, benzyl, benzyloxycarbonyl, tert-butoxycarbonyl,silyl groups (e.g., tert-butyldimethylsilyl),9-fluorenylmethoxycarbonyl, 9-phenyl-9-fluorenyl and arylsulfonyl groups(e.g., toluenesulfonyl). Exemplary oxygen protecting groups include, butare not limited to, allyloxycarbonyl, benzoyl, benzyl, tert-butyl, silylgroups (e.g., tert-butyldimethylsilyl), 2-ethoxyethyl, p-methoxybenzyl,methoxymethyl, pivaloyl, tetrahydropyran-2-yl and trityl. Exemplarycarboxyl protecting groups include, without limitation, methyl, allyl,benzyl, silyl groups (e.g., tert-butyldimethylsilyl) and p-nitrobenzyl.Exemplary carbonyl protecting groups include, but are not limited to,acetyl groups (e.g., O,O-acetals).

[0120] Protecting groups may be removed using methods known in theliterature. For example, for the removal of nitrogen protecting groupssee Greene et al. (1991) Protecting Groups in Organic Synthesis, 2^(nd)ed., John Wiley & Sons, New York, pp. 309-405 and Kocienski (1994)Protecting Groups, Thieme, New York, pp. 185-243. Methods for theremoval of particular protecting groups are exemplified herein.

Utility

[0121] The PKs whose catalytic activity is modulated by the compounds ofthis invention include protein tyrosine kinases of which there are twotypes, receptor tyrosine kinases (RTKs) and cellular tyrosine kinases(CTKs), and serine-threonine kinases (STKs). RTK mediated signaltransduction, is initiated by extracellular interaction with a specificgrowth factor (ligand), followed by receptor dimerization, transientstimulation of the intrinsic protein tyrosine kinase activity andphosphorylation. Binding sites are thereby created for intracellularsignal transduction molecules and lead to the formation of complexeswith a spectrum of cytoplasmic signaling molecules that facilitate theappropriate cellular response (e.g., cell division, metabolic effects onthe extracellular microenvironment, etc.). See, Schlessinger andUllrich, 1992, Neuron 9:303-391.

[0122] It has been shown that tyrosine phosphorylation sites on growthfactor receptors function as high-affinity binding sites for SH2 (srchomology) domains of signaling molecules. Fantl et al., 1992, Cell69:413-423, Songyang et al., 1994, Mol. Cell. Biol. 14:2777-2785),Songyang et al., 1993, Cell 72:767-778, and Koch et al., 1991, Science252:668-678. Several intracellular substrate proteins that associatewith RTKs have been identified. They may be divided into two principalgroups: (1) substrates that have a catalytic domain, and (2) substrateswhich lack such domain but which serve as adapters and associate withcatalytically active molecules. Songyang et al., 1993, Cell 72:767-778.The specificity of the interactions between receptors and SH2 domains oftheir substrates is determined by the amino acid residues immediatelysurrounding the phosphorylated tyrosine residue. Differences in thebinding affinities between SH2 domains and the amino acid sequencessurrounding the phosphotyrosine residues on particular receptors areconsistent with the observed differences in their substratephosphorylation profiles. Songyang et al., 1993, Cell 72:767-778. Theseobservations suggest that the function of each RTK is determined notonly by its pattern of expression and ligand availability but also bythe array of downstream signal transduction pathways that are activatedby a particular receptor. Thus, phosphorylation provides an importantregulatory step which determines the selectivity of signaling pathwaysrecruited by specific growth factor receptors, as well asdifferentiation factor receptors.

[0123] STKs, being primarily cytosolic, affect the internal biochemistryof the cell, often as a down-line response to a PTK event. STKs havebeen implicated in the signaling process which initiates DNA synthesisand subsequent mitosis leading to cell proliferation.

[0124] Thus, PK signal transduction results in, among other responses,cell proliferation, differentiation, growth and metabolism. Abnormalcell proliferation may result in a wide array of disorders and diseases,including the development of neoplasia such as carcinoma, sarcoma,glioblastoma and hemangioma, disorders such as leukemia, psoriasis,arteriosclerosis, arthritis and diabetic retinopathy and other disordersrelated to uncontrolled angiogenesis and/or vasculogenesis.

[0125] In another aspect, the protein kinase, the catalytic activity ofwhich is modulated by contact with a compound of this invention, is aprotein tyrosine kinase, more particularly, a receptor protein tyrosinekinase. Among the receptor protein tyrosine kinases whose catalyticactivity can be modulated with a compound of this invention, or saltthereof, are, without limitation, EGF, HER2, HER3, HER4, IR, IGF-1R,IRR, PDGFRα, PDGFRβ, CSFIR, C-Kit, C-fms, Flk-1R, Flk4, KDR/Flk-1,Flt-1, FGFR-1R, FGFR-2R, FGFR-3R and FGFR-4R.

[0126] The protein tyrosine kinase whose catalytic activity is modulatedby contact with a compound of this invention, or a salt thereof, canalso be a non-receptor or cellular protein tyrosine kinase (CTK). Thus,the catalytic activity of CTKs such as, without limitation, Src, Frk,Btk, Csk, Abl, ZAP70, Fes, Fps, Fak, Jak, Ack, Yes, Fyn, Lyn, Lck, Blk,Hck, Fgr and Yrk, may be modulated by contact with a compound or salt ofthis invention.

[0127] Still another group of PKs which may have their catalyticactivity modulated by contact with a compound of this invention are theserine-threonine protein kinases such as, without limitation, CDK2 andRaf.

[0128] In another aspect, this invention relates to a method fortreating or preventing a PK related disorder by administering atherapeutically effective amount of a compound of this invention, or asalt thereof, to an organism.

[0129] It is also an aspect of this invention that a pharmaceuticalcomposition containing a compound of this invention, or a salt thereof,is administered to an organism for the purpose of preventing or treatinga PK related disorder.

[0130] This invention is therefore directed to compounds that modulatePK signal transduction by affecting the enzymatic activity of RTKs, CTKsand/or STKs, thereby interfering with the signals transduced by suchproteins. More particularly, the present invention is directed tocompounds which modulate RTK, CTK and/or STK mediated signaltransduction pathways as a therapeutic approach to cure many kinds ofsolid tumors, including but not limited to carcinomas, sarcomasincluding Kaposi's sarcoma, erythroblastoma, glioblastoma, meningioma,astrocytoma, melanoma and myoblastoma. Treatment or prevention ofnon-solid tumor cancers such as leukemia are also contemplated by thisinvention. Indications may include, but are not limited to braincancers, bladder cancers, ovarian cancers, gastric cancers, pancreaticcancers, colon cancers, blood cancers, lung cancers and bone cancers.

[0131] Further examples, without limitation, of the types of disordersrelated to inappropriate PK activity that the compounds described hereinmay be useful in preventing, treating and studying, are cellproliferative disorders, fibrotic disorders, metabolic disorders andinfectious diseases.

[0132] Cell proliferative disorders, which may be prevented, treated orfurther studied by the present invention include cancer, blood vesselproliferative disorders and mesangial cell proliferative disorders.

[0133] Blood vessel proliferative disorders refer to disorders relatedto abnormal vasculogenesis (blood vessel formation) and angiogenesis(spreading of blood vessels). While vasculogenesis and angiogenesis playimportant roles in a variety of normal physiological processes such asembryonic development, corpus luteum formation, wound healing and organregeneration, they also play a pivotal role in cancer development wherethey result in the formation of new capillaries needed to keep a tumoralive. Other examples of blood vessel proliferation disorders includearthritis, where new capillary blood vessels invade the joint anddestroy cartilage, and ocular diseases, like diabetic retinopathy, wherenew capillaries in the retina invade the vitreous, bleed and causeblindness.

[0134] Two structurally related RTKs have been identified to bind VEGFwith high affinity: the fms-like tyrosine 1 (fit-1) receptor (Shibuya etal., 1990, Oncogene,5:519-524; De Vries et al., 1992, Science,255:989-991) and the KDR/FLK-1 receptor, also known as VEGF-R2. Vascularendothelial growth factor (VEGF) has been reported to be an endothelialcell specific mitogen with in vitro endothelial cell growth promotingactivity. Ferrara & Henzel, 1989, Biochem. Biophys. Res. Comm.,161:851-858; Vaisman et al., 1990, J. Biol. Chem., 265:19461-19566.Information set forth in U.S. application Ser. Nos. 08/193,829,08/038,596 and 07/975,750, strongly suggest that VEGF is not onlyresponsible for endothelial cell proliferation, but also is the primeregulator of normal and pathological angiogenesis. See generally,Klagsburn & Soker, 1993, Current Biology, 3(10)699-702; Houck, et al.,1992, J. Biol. Chem., 267:26031-26037.

[0135] Normal vasculogenesis and angiogenesis play important roles in avariety of physiological processes such as embryonic development, woundhealing, organ regeneration and female reproductive processes such asfollicle development in the corpus luteum during ovulation and placentalgrowth after pregnancy. Folkman & Shing, 1992, J. Biological Chem.,267(16):10931-34. Uncontrolled vasculogenesis and/or angiogenesis hasbeen associated with diseases such as diabetes as well as with malignantsolid tumors that rely on vascularization for growth. Klagsburn & Soker,1993, Current Biology, 3(10):699-702; Folkham, 1991, J. Natl. CancerInst., 82:4-6; Weidner, et al., 1991, New Engl. J. Med., 324:1-5.

[0136] As presently understood, the role of VEGF in endothelial cellproliferation and migration during angiogenesis and vasculogenesisindicates an important role for the KDR/FLK-1 receptor in theseprocesses. Diseases such as diabetes mellitus (Folkman, 198, in XIthCongress of Thrombosis and Haemostasis (Verstraeta, et al., eds.), pp.583-596, Leuven University Press, Leuven) and arthritis, as well asmalignant tumor growth may result from uncontrolled angiogenesis. Seee.g., Folkman, 1971, N. Engl. J. Med., 285:1182-1186. The receptors towhich VEGF specifically binds are an important and powerful therapeutictarget for the regulation and modulation of vasculogenesis and/orangiogenesis and a variety of severe diseases which involve abnormalcellular growth caused by such processes. Plowman, et al., 1994, DN&P,7(6):334-339. More particularly, the KDR/FLK-1 receptor's highlyspecific role in neovascularization make it a choice target fortherapeutic approaches to the treatment of cancer and other diseaseswhich involve the uncontrolled formation of blood vessels.

[0137] Thus, one aspect of the present invention relates to compoundscapable of regulating and/or modulating tyrosine kinase signaltransduction including KDR/FLK-1 receptor signal transduction in orderto inhibit or promote angiogenesis and/or vasculogenesis, that is,compounds that inhibit, prevent, or interfere with the signal transducedby KDR/FLK-1 when activated by ligands such as VEGF. Although it isbelieved that the compounds of the present invention act on a receptoror other component along the tyrosine kinase signal transductionpathway, they may also act directly on the tumor cells that result fromuncontrolled angiogenesis.

[0138] Although the nomenclature of the human and murine counterparts ofthe generic “flk-I” receptor differ, they are, in many respects,interchangeable. The murine receptor, Flk-1, and its human counterpart,KDR, share a sequence homology of 93.4% within the intracellular domain.Likewise, murine FLK-I binds human VEGF with the same affinity as mouseVEGF, and accordingly, is activated by the ligand derived from eitherspecies. Millauer et al., 1993, Cell, 72:835-846; Quinn et al., 1993,Proc. Natl. Acad. Sci. USA, 90:7533-7537. FLK-1 also associates with andsubsequently tyrosine phosphorylates human RTK substrates (e.g., PLC-γor p85) when co-expressed in 293 cells (human embryonal kidneyfibroblasts).

[0139] Models which rely upon the FLK-1 receptor therefore are directlyapplicable to understanding the KDR receptor. For example, use of themurine FLK-1 receptor in methods which identify compounds that regulatethe murine signal transduction pathway are directly applicable to theidentification of compounds which may be used to regulate the humansignal transduction pathway, that is, which regulate activity related tothe KDR receptor. Thus, chemical compounds identified as inhibitors ofKDR/FLK-1 in vitro, can be confirmed in suitable in vivo models. Both invivo mouse and rat animal models have been demonstrated to be ofexcellent value for the examination of the clinical potential of agentsacting on the KDR/FLK-1 induced signal transduction pathway.

[0140] Thus, in one aspect, this invention is directed to compounds thatregulate, modulate and/or inhibit vasculogenesis and/or angiogenesis byaffecting the enzymatic activity of the KDR/FLK-1 receptor andinterfering with the signal transduced by KDR/FLK-1. In another aspect,the present invention is directed to compounds which regulate, modulateand/or inhibit the KDR/FLK-1 mediated signal transduction pathway as atherapeutic approach to the treatment of many kinds of solid tumorsincluding, but not limited to, glioblastoma, melanoma and Kaposi'ssarcoma, and ovarian, lung, mammary, prostate, pancreatic, colon andepidermoid carcinoma. In addition, data suggest the administration ofcompounds which inhibit the KDR/Flk-1 mediated signal transductionpathway may also be used in the treatment of hemangioma, restenois anddiabetic retinopathy.

[0141] A further aspect of this invention relates to the inhibition ofvasculogenesis and angiogenesis by other receptor-mediated pathways,including the pathway comprising the flt-1 receptor.

[0142] Receptor tyrosine kinase mediated signal transduction isinitiated by extracellular interaction with a specific growth factor(ligand), followed by receptor dimerization, transient stimulation ofthe intrinsic protein tyrosine kinase activity and autophosphorylation.Binding sites are thereby created for intracellular signal transductionmolecules which leads to the formation of complexes with a spectrum ofcytoplasmic signaling molecules that facilitate the appropriate cellularresponse, e.g., cell division and metabolic effects to the extracellularmicroenvironment. See, Schlessinger and Ullrich, 1992, Neuron, 9:1-20.

[0143] The close homology of the intracellular regions of KDR/FLK-1 withthat of the PDGF-β receptor (50.3% homology) and/or the related flt-1receptor indicates the induction of overlapping signal transductionpathways. For example, for the PDGF-β receptor, members of the srcfamily (Twamley et al., 1993, Proc. Natl. Acad. Sci. USA, 90:7696-7700),phosphatidylinositol-3′-kinase (Hu et al., 1992, Mol. Cell. Biol.,12:981-990), phospholipase cγ (Kashishian & Cooper, 1993, Mol. Cell.Biol., 4:49-51), ras-GTPase-activating protein, (Kashishian et al.,1992, EMBO J., 11:1373-1382), PTP-ID/syp (Kazlauskas et al., 1993, Proc.Natl. Acad. Sci. USA, 10 90:6939-6943), Grb2 (Arvidsson et al., 1994,Mol. Cell. Biol., 14:6715-6726), and the adapter molecules She and Nck(Nishimura et al., 1993, Mol. Cell. Biol., 13:6889-6896), have beenshown to bind to regions involving different autophosphorylation sites.See generally, Claesson-Welsh, 1994, Prog. Growth Factor Res., 5:37-54.Thus, it is likely that signal transduction pathways activated byKDR/FLK-1 include the ras pathway (Rozakis et al., 1992, Nature,360:689-692), the PI-3′-kinase, the src-mediated and the plcγ-mediatedpathways. Each of these pathways may play a critical role in theangiogenic and/or vasculogenic effect of KDR/FLK-1 in endothelial cells.Consequently, a still further aspect of this invention relates to theuse of the organic compounds described herein to modulate angiogenesisand vasculogenesis as such processes are controlled by these pathways.

[0144] Conversely, disorders related to the shrinkage, contraction orclosing of blood vessels, such as restenosis, are also implicated andmay be treated or prevented by the methods of this invention.

[0145] Fibrotic disorders refer to the abnormal formation ofextracellular matrices. Examples of fibrotic disorders include hepaticcirrhosis and mesangial cell proliferative disorders. Hepatic cirrhosisis characterized by the increase in extracellular matrix constituentsresulting in the formation of a hepatic scar. An increased extracellularmatrix resulting in a hepatic scar can also be caused by a viralinfection such as hepatitis. Lipocytes appear to play a major role inhepatic cirrhosis. Other fibrotic disorders implicated includeatherosclerosis.

[0146] Mesangial cell proliferative disorders refer to disorders broughtabout by abnormal proliferation of mesangial cells. Mesangialproliferative disorders include various human renal diseases such asglomerulonephritis, diabetic nephropathy and malignant nephrosclerosisas well as such disorders as thrombotic microangiopathy syndromes,transplant rejection, and glomerulopathies. The RTK PDGFR has beenimplicated in the maintenance of mesangial cell proliferation. Floege etal., 1993, Kidney International 43:47S-54S.

[0147] Many cancers are cell proliferative disorders and, as notedpreviously, PKs have been associated with cell proliferative disorders.Thus, it is not surprising that PKs such as, for example, members of theRTK family have been associated with the development of cancer. Some ofthese receptors, like EGFR (Tuzi et al., 1991, Br. J. Cancer 63:227-233,Torp et al., 1992, APMIS 100:713-719) HER2/neu (Slamon et al., 1989,Science 244:707-712) and PDGF-R (Kumabe et al., 1992, Oncogene,7:627-633) are over-expressed in many tumors and/or persistentlyactivated by autocrine loops. In fact, in the most common and severecancers these receptor over-expressions (Akbasak and Suner-Akbasak etal., 1992, J. Neurol. Sci., 111:119-133, Dickson et al., 1992, CancerTreatment Res. 61:249-273, Korc et al., 1992, J. Clin. Invest.90:1352-1360) and autocrine loops (Lee and Donoghue, 1992, J. Cell.Biol., 118:1057-1070, Korc et al., supra, Akbasak and Suner-Akbasak etal., supra) have been demonstrated. For example, EGFR has beenassociated with squamous cell carcinoma, astrocytoma, glioblastoma, headand neck cancer, lung cancer and bladder cancer. HER2 has beenassociated with breast, ovarian, gastric, lung, pancreatic and bladdercancer. PDGFR has been associated with glioblastoma and melanoma as wellas lung, ovarian and prostate cancer. The RTK c-met has also beenassociated with malignant tumor formation. For example, c-met has beenassociated with, among other cancers, colorectal, thyroid, pancreatic,gastric and hepatocellular carcinomas and lymphomas. Additionally c-methas been linked to leukemia. Over-expression of the c-met gene has alsobeen detected in patients with Hodgkins disease and Burkitts disease.

[0148] IGF-IR, in addition to being implicated in nutritional supportand in type-II diabetes, has also been associated with several types ofcancers. For example, IGF-I has been implicated as an autocrine growthstimulator for several tumor types, e.g. human breast cancer carcinomacells (Arteaga et al., 1989, J. Clin. Invest. 84:1418-1423) and smalllung tumor cells (Macauley et al., 1990, Cancer Res., 50:2511-2517). Inaddition, IGF-I, while integrally involved in the normal growth anddifferentiation of the nervous system, also appears to be an autocrinestimulator of human gliomas. Sandberg-Nordqvist et al., 1993, CancerRes. 53:2475-2478. The importance of IGF-IR and its ligands in cellproliferation is further supported by the fact that many cell types inculture (fibroblasts, epithelial cells, smooth muscle cells,T-lymphocytes, myeloid cells, chondrocytes and osteoblasts (the stemcells of the bone marrow)) are stimulated to grow by IGF-I. Goldring andGoldring, 1991, Eukaryotic Gene Expression,1:301-326. In a series ofrecent publications, Baserga suggests that IGF-IR plays a central rolein the mechanism of transformation and, as such, could be a preferredtarget for therapeutic interventions for a broad spectrum of humanmalignancies. Baserga, 1995, Cancer Res., 55:249-252, Baserga, 1994,Cell 79:927-930, Coppola et al., 1994, Mol. Cell. Biol., 14:4588-4595.

[0149] STKs have been implicated in many types of cancer including,notably, breast cancer (Cance, et al., Int. J. Cancer, 54:571-77(1993)).

[0150] The association between abnormal PK activity and disease is notrestricted to cancer. For example, RTKs have been associated withdiseases such as psoriasis, diabetes mellitus, endometriosis,angiogenesis, atheromatous plaque development, Alzheimer's disease, vonHippel-Lindau disease, epidermal hyperproliferation, neurodegenerativediseases, age-related macular degeneration and hemangiomas. For example,EGFR has been indicated in corneal and dermal wound healing. Defects inInsulin-R and IGF-1R are indicated in type-II diabetes mellitus. A morecomplete correlation between specific RTKs and their therapeuticindications is set forth in Plowman et al., 1994, DN&P 7:334-339.

[0151] As noted previously, CTKs including, but not limited to, src,abl, fps, yes, fyn, lyn, lck, blk, hck, fgr and yrk (reviewed by Bolenet al., 1992, FASEB J., 6:3403-3409), are also involved in theproliferative and metabolic signal transduction pathway and thus couldbe expected, and have been shown, to be involved in many PTK-mediateddisorders to which the present invention is directed. For example,mutated src (v-src) has been shown to be an oncoprotein (pp60^(v-src))in chicken. Moreover, its cellular homolog, the proto-oncogenepp60^(c-src) transmits oncogenic signals of many receptors.Over-expression of EGFR or HER2/neu in tumors leads to the constitutiveactivation of pp60^(c-src), which is characteristic of malignant cellsbut absent in normal cells. On the other hand, mice deficient in theexpression of c-src exhibit an osteopetrotic phenotype, indicating a keyparticipation of c-src in osteoclast function and a possible involvementin related disorders.

[0152] Similarly, Zap70 has been implicated in T-cell signaling whichmay relate to autoimmune disorders.

[0153] STKs have been associated with inflammation, autoimmune disease,immunoresponses, and hyperproliferation disorders such as restenosis,fibrosis, psoriasis, osteoarthritis and rheumatoid arthritis.

[0154] PKs have also been implicated in embryo implantation. Thus, thecompounds of this invention may provide an effective method ofpreventing such embryo implantation and thereby be useful as birthcontrol agents.

[0155] In yet another aspect, the compounds of the instant invention canalso be used as anti-infective agents. For example, indolinone compoundsare known to exhibit antibacterial and antifungal activities. See, e.g.,Singh and Jha (1989) “Indolinone derivatives as potential antimicrobialagents,” Zentralbl. Mikrobiol. 144(2):105-109. In addition, indolinonecompounds have been reported to exhibit significant antiviral activity.See, e.g., Maass et al. (1993) “Viral resistance to thethiazolo-iso-indolinones, a new class of nonnucleoside inhibitors ofhuman immunodeficiency virus type 1 reverse transcriptase,” Antimicrob.Agents Chemother. 37(12):2612-2617.

[0156] Finally, both RTKs and CTKs are currently suspected as beinginvolved in hyperimmune disorders.

[0157] A method for identifying a chemical compound that modulates thecatalytic activity of one or more of the above discussed protein kinasesis another aspect of this invention. The method involves contactingcells expressing the desired protein kinase with a compound of thisinvention (or its salt) and monitoring the cells for any effect that thecompound has on them. The effect may be any observable, either to thenaked eye or through the use of instrumentation, change or absence ofchange in a cell phenotype. The change or absence of change in the cellphenotype monitored may be, for example, without limitation, a change orabsence of change in the catalytic activity of the protein kinase in thecells or a change or absence of change in the interaction of the proteinkinase with a natural binding partner.

Pharmaceutical Compositions and Administration

[0158] A compound of the present invention or a physiologicallyacceptable salt thereof, can be administered as such to a human patientor can be administered in pharmaceutical compositions in which theforegoing materials are mixed with suitable carriers or excipient(s).Techniques for formulation and administration of drugs may be found in“Remington's Pharmacological Sciences,” Mack Publishing Co., Easton, PA,latest edition.

Routes of Administration

[0159] As used herein, “administer” or “administration” refers to thedelivery of a compound or salt of the present invention or of apharmaceutical composition containing a compound or salt of thisinvention to an organism for the purpose of prevention or treatment of aPK-related disorder.

[0160] Suitable routes of administration may include, withoutlimitation, oral, rectal, transmucosal or intestinal administration orintramuscular, subcutaneous, intramedullary, intrathecal, directintraventricular, intravenous, intravitreal, intraperitoneal,intranasal, or intraocular injections. The preferred routes ofadministration are oral and parenteral.

[0161] Alternatively, one may administer the compound in a local ratherthan systemic manner, for example, via injection of the compounddirectly into a solid tumor, often in a depot or sustained releaseformulation.

[0162] Furthermore, one may administer the drug in a targeted drugdelivery system, for example, in a liposome coated with tumor-specificantibody. The liposomes will be targeted to and taken up selectively bythe tumor.

Composition/Formulation

[0163] Pharmaceutical compositions of the present invention may bemanufactured by processes well known in the art, e.g., by means ofconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping, lyophilizing processes or spraydrying.

[0164] Pharmaceutical compositions for use in accordance with thepresent invention may be formulated in conventional manner using one ormore physiologically acceptable carriers comprising excipients andauxiliaries which facilitate processing of the active compounds intopreparations which can be used pharmaceutically. Proper formulation isdependent upon the route of administration chosen.

[0165] For injection, the compounds of the invention may be formulatedin aqueous solutions, preferably in physiologically compatible bufferssuch buffers with or without a low concentration of surfactant orcosolvent, or physiological saline buffer. For transmucosaladministration, penetrants appropriate to the barrier to be permeatedare used in the formulation. Such penetrants are generally known in theart.

[0166] For oral administration, the compounds can be formulated bycombining the active compounds with pharmaceutically acceptable carrierswell known in the art. Such carriers enable the compounds of theinvention to be formulated as tablets, pills, lozenges, dragees,capsules, liquids, gels, syrups, slurries, suspensions and the like, fororal ingestion by a patient. Pharmaceutical preparations for oral usecan be made using a solid excipient, optionally grinding the resultingmixture, and processing the mixture of granules, after adding othersuitable auxiliaries if desired, to obtain tablets or dragee cores.Useful excipients are, in particular, fillers such as sugars, includinglactose, sucrose, mannitol, or sorbitol, cellulose preparations such as,for example, maize starch, wheat starch, rice starch and potato starchand other materials such as gelatin, gum tragacanth, methyl cellulose,hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/orpolyvinyl-pyrrolidone (PVP). If desired, disintegrating agents may beadded, such as cross-linked polyvinyl pyrrolidone, agar, or alginicacid. A salt such as sodium alginate may also be used.

[0167] Dragee cores are provided with suitable coatings. For thispurpose, concentrated sugar solutions may be used which may optionallycontain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel,polyethylene glycol, and/or titanium dioxide, lacquer solutions, andsuitable organic solvents or solvent mixtures. Dyestuffs or pigments maybe added to the tablets or dragee coatings for identification or tocharacterize different combinations of active compound doses.

[0168] Pharmaceutical compositions which can be used orally includepush-fit capsules made of gelatin, as well as soft, sealed capsules madeof gelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules can contain the active ingredients in admixture with a fillersuch as lactose, a binder such as starch, and/or a lubricant such astalc or magnesium stearate and, optionally, stabilizers. In softcapsules, the active compounds may be dissolved or suspended in suitableliquids, such as fatty oils, liquid paraffin, liquid polyethyleneglycols, cremophor, capmul, medium or long chain mono- di- ortriglycerides. Stabilizers may be added in these formulations, also.

[0169] For administration by inhalation, the compounds for use accordingto the present invention are conveniently delivered in the form of anaerosol spray using a pressurized pack or a nebulizer and a suitablepropellant, e.g., without limitation, dichlorodifluoromethane,trichlorofluoromethane, dichlorotetra-fluoroethane or carbon dioxide. Inthe case of a pressurized aerosol, the dosage unit may be controlled byproviding a valve to deliver a metered amount. Capsules and cartridgesof, for example, gelatin for use in an inhaler or insufflator may beformulated containing a powder mix of the compound and a suitable powderbase such as lactose or starch.

[0170] The compounds may also be formulated for parenteraladministration, e.g., by bolus injection or continuous infusion.Formulations for injection may be presented in unit dosage form, e.g.,in ampoules or in multi-dose containers, with an added preservative. Thecompositions may take such forms as suspensions, solutions or emulsionsin oily or aqueous vehicles, and may contain formulating materials suchas suspending, stabilizing and/or dispersing agents.

[0171] Pharmaceutical compositions for parenteral administration includeaqueous solutions of a water soluble form, such as, without limitation,a salt, of the active compound. Additionally, suspensions of the activecompounds may be prepared in a lipophilic vehicle. Suitable lipophilicvehicles include fatty oils such as sesame oil, synthetic fatty acidesters such as ethyl oleate and triglycerides, or materials such asliposomes. Aqueous injection suspensions may contain substances whichincrease the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, or dextran. Optionally, the suspension may alsocontain suitable stabilizers and/or agents that increase the solubilityof the compounds to allow for the preparation of highly concentratedsolutions.

[0172] Alternatively, the active ingredient may be in powder form forconstitution with a suitable vehicle, e.g., sterile, pyrogen-free waterwith or without additional sufactants or cosolvents such as polysorbate80, Cremophor, cyclodextrin sulfobutyl ether, propylene glycol orpolyethylene glycol such as PEG-300 or PEG-400, before use.

[0173] The compounds may also be formulated in rectal compositions suchas suppositories or retention enemas, using, e.g., conventionalsuppository bases such as cocoa butter or other glycerides.

[0174] In addition to the formulations described previously, thecompounds may also be formulated as depot preparations. Such long actingformulations may be administered by implantation (for example,subcutaneously or intramuscularly) or by intramuscular injection. Acompound of this invention may be formulated for this route ofadministration with suitable polymeric or hydrophobic materials (forinstance, in an emulsion with a pharmacologically acceptable oil), withion exchange resins, or as a sparingly soluble derivative such as,without limitation, a sparingly soluble salt.

[0175] A non-limiting example of a pharmaceutical carrier for thehydrophobic compounds of the invention is a cosolvent system comprisingbenzyl alcohol, a nonpolar surfactant, a water-miscible organic polymerand an aqueous phase such as the VPD co-solvent system. VPD is asolution of 3% w/v benzyl alcohol, 8% w/v of the nonpolar surfactantPolysorbate 80™, and 65% w/v polyethylene glycol 300, made up to volumein absolute ethanol. The VPD co-solvent system (VPD:D5W) consists of VPDdiluted 1:1 with a 5% dextrose in water solution. This co-solvent systemdissolves hydrophobic compounds well, and itself produces low toxicityupon systemic administration. Naturally, the proportions of such aco-solvent system may be varied considerably without destroying itssolubility and toxicity characteristics. Furthermore, the identity ofthe co-solvent components may be varied: for example, other low-toxicitynonpolar surfactants may be used instead of Polysorbate 80™, thefraction size of polyethylene glycol may be varied, other biocompatiblepolymers may replace polyethylene glycol, e.g., polyvinyl pyrrolidone,and other sugars or polysaccharides may substitute for dextrose.

[0176] Alternatively, other delivery systems for hydrophobicpharmaceutical compounds may be employed. Liposomes and emulsions arewell known examples of delivery vehicles or carriers for hydrophobicdrugs. In addition, certain organic solvents such as dimethylsulfoxidealso may be employed, although often at the cost of greater toxicity.

[0177] Additionally, the compounds may be delivered using asustained-release system, such as semipermeable matrices of solidhydrophobic polymers containing the therapeutic agent. Varioussustained-release materials have been established and are well known bythose skilled in the art. Sustained-release capsules may, depending ontheir chemical nature, release the compounds for a few weeks up to over100 days. Depending on the chemical nature and the biological stabilityof the therapeutic reagent, additional strategies for proteinstabilization may be employed.

[0178] The pharmaceutical compositions herein also may comprise suitablesolid or gel phase carriers or excipients. Examples of such carriers orexcipients include, but are not limited to, calcium carbonate, calciumphosphate, various sugars, starches, cellulose derivatives, gelatin, andpolymers such as polyethylene glycols.

[0179] Many of the PK modulating compounds of the invention may beprovided as physiologically acceptable salts wherein the claimedcompound may form the negatively or the positively charged species.Examples of salts in which the compound forms the positively chargedmoiety include, without limitation, quaternary ammonium (definedelsewhere herein), salts such as the hydrochloride, sulfate, citrate,mesylate, lactate, tartrate, maleate, succinate wherein the nitrogenatom of the quaternary ammonium group is a nitrogen of the selectedcompound of this invention which has reacted with the appropriate acid.Salts in which a compound of this invention forms the negatively chargedspecies include, without limitation, the sodium, potassium, calcium andmagnesium salts formed by the reaction of a carboxylic acid group in thecompound with an appropriate base (e.g. sodium hydroxide (NaOH),potassium hydroxide (KOH), Calcium hydroxide (Ca(OH)₂), etc.).

Dosage

[0180] Pharmaceutical compositions suitable for use in the presentinvention include compositions wherein the active ingredients arecontained in an amount sufficient to achieve the intended purpose, i.e.,the modulation of PK activity or the treatment or prevention of aPK-related disorder.

[0181] More specifically, a therapeutically effective amount means anamount of compound effective to prevent, alleviate or amelioratesymptoms of disease or prolong the survival of the subject beingtreated. Therapeutically effective amounts of compounds of Formula I mayrange from approximately 10 mg/m² to 400 mg/m², preferably 50 mg/m² to300 mg/m², more preferably 100 mg/m² to 220 mg/m², even more preferably195 mg/m².

[0182] For any compound used in the methods of the invention, thetherapeutically effective amount or dose can be estimated initially fromcell culture assays. Then, the dosage can be formulated for use inanimal models so as to achieve a circulating concentration range thatincludes the IC₅₀ as determined in cell culture (i.e., the concentrationof the test compound which achieves a half-maximal inhibition of the PKactivity). Such information can then be used to more accuratelydetermine useful doses in humans.

[0183] Toxicity and therapeutic efficacy of the compounds describedherein can be determined by standard pharmaceutical procedures in cellcultures or experimental animals, e.g., by determining the IC₅₀ and theLD₅₀ (both of which are discussed elsewhere herein) for a subjectcompound. The data obtained from these cell culture assays and animalstudies can be used in formulating a range of dosage for use in humans.The dosage may vary depending upon the dosage form employed and theroute of administration utilized. The exact formulation, route ofadministration and dosage can be chosen by the individual physician inview of the patient's condition. (See e.g., Fingl, et al., 1975, in “ThePharmacological Basis of Therapeutics”, Ch. 1 p.1).

[0184] Dosage amount and interval may be adjusted individually toprovide plasma levels of the active species which are sufficient tomaintain the kinase modulating effects. These plasma levels are referredto as minimal effective concentrations (MECs). The MEC will vary foreach compound but can be estimated from in vitro data, e.g., theconcentration necessary to achieve 50-90% inhibition of a kinase may beascertained using the assays described herein. Preferably, the Dosagesnecessary to achieve the MEC will depend on individual characteristicsand route of administration. HPLC assays or bioassays can be used todetermine plasma concentrations.

[0185] Dosage intervals can also be determined using MEC value.Compounds should be administered using a regimen that maintains plasmalevels above the MEC for 10-90% of the time, preferably between 30-90%and most preferably between 50-90%. In cases of local administration orselective uptake, the effective local concentration of the drug may notbe related to plasma concentration and other procedures known in the artmay be employed to determine the correct dosage amount and interval.

[0186] The amount of a composition administered will, of course, bedependent on the subject being treated, the severity of the affliction,the manner of administration, the judgment of the prescribing physician,etc.

Packaging

[0187] The compositions may, if desired, be presented in a pack ordispenser device, such as an FDA approved kit, which may contain one ormore unit dosage forms containing the active ingredient. The pack mayfor example comprise metal or plastic foil, such as a blister pack. Thepack or dispenser device may be accompanied by instructions foradministration. The pack or dispenser may also be accompanied by anotice associated with the container in a form prescribed by agovernmental agency regulating the manufacture, use or sale ofpharmaceuticals, which notice is reflective of approval by the agency ofthe form of the compositions or of human or veterinary administration.Such notice, for example, may be of the labeling approved by the U.S.Food and Drug Administration for prescription drugs or of an approvedproduct insert. Compositions comprising a compound of the inventionformulated in a compatible pharmaceutical carrier may also be prepared,placed in an appropriate container, and labeled for treatment of anindicated condition. Suitable conditions indicated on the label mayinclude treatment of a tumor, inhibition of angiogenesis, treatment offibrosis, diabetes, and the like.

[0188] It is also an aspect of this invention that a compound describedherein, or its salt, might be combined with other chemotherapeuticagents for the treatment of the diseases and disorders discussed above.For instance, a compound or salt of this invention might be combinedwith alkylating agents such as fluorouracil (5-FU) alone or in furthercombination with leukovorin; or other alkylating agents such as, withoutlimitation, other pyrimidine analogs such as UFT, capecitabine,gemcitabine and cytarabine, the alkyl sulfonates, e.g., busulfan (usedin the treatment of chronic granulocytic leukemia), improsulfan andpiposulfan; aziridines, e.g., benzodepa, carboquone, meturedepa anduredepa; ethyleneimines and methylmelamines, e.g., altretamine,triethylenemelamine, triethylenephosphoramide,triethylenethiophosphoramide and trimethylolmelamine; and the nitrogenmustards, e.g., chlorambucil (used in the treatment of chroniclymphocytic leukemia, primary macroglobulinemia and non-Hodgkin'slymphoma), cyclophosphamide (used in the treatment of Hodgkin's disease,multiple myeloma, neuroblastoma, breast cancer, ovarian cancer, lungcancer, Wilm's tumor and rhabdomyosarcoma), estramustine, ifosfamide,novembrichin, prednimustine and uracil mustard (used in the treatment ofprimary thrombocytosis, non-Hodgkin's lymphoma, Hodgkin's disease andovarian cancer); and triazines, e.g., dacarbazine (used in the treatmentof soft tissue sarcoma).

[0189] Likewise a compound or salt of this invention might be expectedto have a beneficial effect in combination with other antimetabolitechemotherapeutic agents such as, without limitation, folic acid analogs,e.g. methotrexate (used in the treatment of acute lymphocytic leukemia,choriocarcinoma, mycosis fungiodes breast cancer, head and neck cancerand osteogenic sarcoma) and pteropterin; and the purine analogs such asmercaptopurine and thioguanine which find use in the treatment of acutegranulocytic, acute lymphocytic and chronic granulocytic leukemias.

[0190] A compound or salt of this invention might also be expected toprove efficacious in combination with natural product basedchemotherapeutic agents such as, without limitation, the vincaalkaloids, e.g., vinblastin (used in the treatment of breast andtesticular cancer), vincristine and vindesine; the epipodophylotoxins,e.g., etoposide and teniposide, both of which are useful in thetreatment of testicular cancer and Kaposi's sarcoma; the antibioticchemotherapeutic agents, e.g., daunorubicin, doxorubicin, epirubicin,mitomycin (used to treat stomach, cervix, colon, breast, bladder andpancreatic cancer), dactinomycin, temozolomide, plicamycin, bleomycin(used in the treatment of skin, esophagus and genitourinary tractcancer); and the enzymatic chemotherapeutic agents such asL-asparaginase.

[0191] In addition to the above, a compound or salt of this inventionmight be expected to have a beneficial effect used in combination withthe platinum coordination complexes (cisplatin, etc.); substituted ureassuch as hydroxyurea; methylhydrazine derivatives, e.g., procarbazine;adrenocortical suppressants, e.g., mitotane, aminoglutethimide; andhormone and hormone antagonists such as the adrenocorticosteriods (e.g.,prednisone), progestins (e.g., hydroxyprogesterone caproate); estrogens(e.g., diethylstilbesterol); antiestrogens such as tamoxifen; androgens,e.g., testosterone propionate; and aromatase inhibitors (such asanastrozole).

[0192] Finally, the combination of a compound of this invention might beexpected to be particularly effective in combination with CAMPTOSAR™,GLEEVEC™, HERCEPTIN™, ENDOSTATIN™, Cox-2 inhibitors, MITOXANTRONE™ orPACLITAXEL™ for the treatment of solid tumor cancers or leukemias suchas, without limitation, acute myelogenous (non-lymphocytic) leukemia.

EXAMPLES

[0193] The following preparations and examples are given to enable thoseskilled in the art to more clearly understand and to practice thepresent invention. They should not be considered as limiting the scopeof the invention, but merely as being illustrative and representativethereof.

[0194] In general HPLC data was obtained with a Zorbax SB C18 column(4.6 mm ID×7.5 cm), a Perkin Elmer series 200 pump programmed to runfrom 10% acetonitrile/water 0.1% TFA (solvent A) to 90%acetonitrile/water (solvent B) with a flow rate of 1.5 mL/min. After 0.1min on solvent A, a 5 min linear program to solvent B was run, followedby 3 min on solvent B, before recycling to solvent A (2 min). Detectionwas with a Perkin Elmer diode array detector recording at 215 and 280nM). NMR spectra were recorded on a Bruker instrument at 300 MHz.

Synthetic Examples Example 1(3Z)-3-[(3,5-Dimethyl-1H-pyrrol-2-yl)-methylidene]-1-(1-pyrrolidinylmethyl)-1,3-dihydro-2H-indol-2-one

[0195] Pyrrolidine (450 mg, 6.3 mmol) was added to a stirred solution ofaqueous formaldehyde (500 mg of 38% solution, 6.0 mmol) and3-(3,5-dimethyl-1H-pyrrol-2-ylmethylidene)-1,3-dihydro-indol-2-one (900mg, 3.8 mmol) in methanol (50 mL). After 15 min., the solution wascooled to 0° C. and the precipitate was filtered off, washed with water,and dried to give 1.08 g of the title compound, mp 129-132° C. HPLC Rt4.87 min. ¹H NMR [(CD₃)₂SO] δ1.65 (m, 4H), 2.32 9s, 3H), 2.34 (s, 3H),2.62 (m, 4H), 472 (s, 2H) 6.07 (d, 1H), 7.00 (m, 1H), 7.15 (m, 2H), 7.61(s, 1H), 7.76 (d, 2H) and 13.1 (br s, 1H). Anal. Calcd for C₂₀H₂₃N₃O: C,74.74; H, 7.21; N, 13.07. Found: C, 74.61; H, 7.25; N, 13.03.

Biological Evaluation

[0196] It will be appreciated that, in any given series of compounds, arange of biological activities will be observed. In its presentlypreferred embodiments, this invention relates to novel1-substituted-3-pyrrolidinyl-2-indolinones capable of generating in vivo3-pyrrolidinyl-2-indolinones capable of modulating, regulating and/orinhibiting protein kinase activity. The following assays may be employedto select those compounds demonstrating the optimal degree of thedesired activity.

Assay Procedures

[0197] The following in vitro assays may be used to determine the levelof activity and effect of the different compounds of the presentinvention on one or more of the PKs. Similar assays can be designedalong the same lines for any PK using techniques well known in the art.

[0198] Several of the assays described herein are performed in an ELISA(Enzyme-Linked Immunosorbent Sandwich Assay) format (Voller, et al.,1980, “Enzyme-Linked Immunosorbent Assay,” Manual of ClinicalImmunology, 2d ed., Rose and Friedman, Am. Soc. Of Microbiology,Washington, D.C., pp. 359-371). The general procedure is as follows: acompound is introduced to cells expressing the test kinase, eithernaturally or recombinantly, for a selected period of time after which,if the test kinase is a receptor, a ligand known to activate thereceptor is added. The cells are lysed and the lysate is transferred tothe wells of an ELISA plate previously coated with a specific antibodyrecognizing the substrate of the enzymatic phosphorylation reaction.Non-substrate components of the cell lysate are washed away and theamount of phosphorylation on the substrate is detected with an antibodyspecifically recognizing phosphotyrosine compared with control cellsthat were not contacted with a test compound.

[0199] The presently preferred protocols for conducting the ELISAexperiments for specific PKs is provided below. However, adaptation ofthese protocols for determining the activity of compounds against otherRTKs, as well as for CTKs and STKs, is well within the scope ofknowledge of those skilled in the art. Other assays described hereinmeasure the amount of DNA made in response to activation of a testkinase, which is a general measure of a proliferative response. Thegeneral procedure for this assay is as follows: a compound is introducedto cells expressing the test kinase, either naturally or recombinantly,for a selected period of time after which, if the test kinase is areceptor, a ligand known to activate the receptor is added. Afterincubation at least overnight, a DNA labeling reagent such as5-bromodeoxyuridine (BrdU) or H³-thymidine is added. The amount oflabeled DNA is detected with either an anti-BrdU antibody or bymeasuring radioactivity and is compared to control cells not contactedwith a test compound.

GST-FLK-1 BIOASSAY

[0200] This assay analyzes the tyrosine kinase activity of GST-Flk1 onpoly(glu-tyr) peptides.

[0201] Materials and Reagents

[0202] 1. Corning 96-well ELISA plates (Corning Catalog No. 25805-96).

[0203] 2. poly(glu-tyr) 4:1, lyophilizate (Sigma Catalog No. P0275), 1mg/ml in sterile PBS.

[0204] 3. PBS Buffer: for 1 L, mix 0.2 g KH₂PO₄, 1.15 g Na_(H)PO₄, 0.2 gKCl and 8 g NaCl in approx. 900 ml dH₂O. When all reagents havedissolved, adjust the pH to 7.2 with HCl. Bring total volume to 1 L withdH₂O.

[0205] 4. PBST Buffer: to 1 L of PBS Buffer, add 1.0 ml Tween-20.

[0206] 5. TBB-Blocking Buffer: for 1 L, mix 1.21 g TRIS, 8.77 g NaCl, 1ml TWEEN-20 in approximately 900 ml dH₂O. Adjust pH to 7.2 with HCl. Add10 g BSA, stir to dissolve. Bring total volume to 1 L with dH₂O. Filterto remove particulate matter.

[0207] 6. 1% BSA in PBS: add 10 g BSA to approx. 990 ml PBS buffer, stirto dissolve. Adjust total volume to 1 L with PBS buffer, filter toremove particulate matter.

[0208] 7. 50 mM Hepes pH 7.5.

[0209] 8. GST-Flk1cd purified from sf9 recombinant baculovirustransformation (SUGEN, Inc.).

[0210] 9. 4% DMSO in dH₂O.

[0211] 10. 10 mM ATP in dH₂O.

[0212] 11. 40 mM MnCl₂

[0213] 12. Kinase Dilution Buffer (KDB): mix 10 ml Hepes (pH 7.5), 1 ml5M NaCl, 40 μL 100 mM sodium orthovanadate and 0.4 ml of 5% BSA in dH₂Owith 88.56 ml dH₂O.

[0214] 13. NUNC 96-well V bottom polypropylene plates, AppliedScientific Catalog # AS-72092

[0215] 14. EDTA: mix 14.12 g ethylenediaminetetraacetic acid (EDTA) withapprox. 70 ml dH₂O. Add 10 N NaOH until EDTA dissolves. Adjust pH to8.0. Adjust total volume to 100 ml with dH₂O.

[0216] 15. 1° and 2° Antibody Dilution Buffer: mix 10 ml of 5% BSA inPBS buffer with 89.5 ml TBST.

[0217] 16. Anti-phosphotyrosine rabbit polyclonal antisera (SUGEN, Inc.)

[0218] 17. Goat anti-rabbit HRP conjugate.

[0219] 18. ABST solution: To approx. 900 ml dH₂O add 19.21 g citric acidand 35.49 g Na₂HPO₄. Adjust pH to 4.0 with phosphoric acid. Add2,2′-Azinobis(3-ethyl- benzthiazoline-6-sulfonic acid (ABTS, Sigma, Cat.No. A-1888, hold for approx. ½ hour, filter.

[0220] 19. 30% Hydrogen Peroxide.

[0221] 20. ABST/H₂O₂: add 3 μl of H₂O₂ to 15 ml of ABST solution.

[0222] 21. 0.2 M HCl.

[0223] Procedure

[0224] 1. Coat Corning 96-well ELISA plates with 2 μg of polyEY in 100μl PBS/well, hold at room temperature for 2 hours or at 4° C. overnight.Cover plates to prevent evaporation.

[0225] 2. Remove unbound liquid from wells by inverting plate. Wash oncewith TBST. Pat the plate on a paper towel to remove excess liquid.

[0226] 3. Add 100 μl of 1% BSA in PBS to each well. Incubate, withshaking, for 1 hr. at room temperature.

[0227] 4. Repeat step 2.

[0228] 5. Soak wells with 50 mM HEPES (pH7.5, 150 μl/well).

[0229] 6. Dilute test compound with dH₂O/4% DMSO to 4 times the desiredfinal assay concentration in 96-well polypropylene plates.

[0230] 7. Add 25 μl diluted test compound to each well of ELISA plate.In control wells, place 25 μl of dH₂O/4% DMSO.

[0231] 8. Dilute GST-Flk1 0.005 μg (5 ng)/well in KDB.

[0232] 9. Add 50 μl of diluted enzyme to each well.

[0233] 10. Add 25 μl 0.5 M EDTA to negative control wells.

[0234] 11. Add 25 μl of 40 mM MnCl₂ with 4×ATP (2 μM) to all wells (100μl final volume, 0.5 μM ATP final concentration in each well).

[0235] 12. Incubate, with shaking, for 15 minutes at room temperature.

[0236] 13. Stop reaction by adding 25 μl of 500 mM EDTA to each well.

[0237] 14. Wash 3× with TBST and pat plate on paper towel to removeexcess liquid.

[0238] 15. Add 100 μl per well anti-phosphotyrosine antisera, 1:10,000dilution in antibody dilution buffer. Incubate, with shaking, for 90min. at room temperature.

[0239] 16. Wash as in step 14.

[0240] 17. Add 100 μl/well of goat anti-rabbit HRP conjugate (1:6,000 inantibody dilution buffer). Incubate, with shaking, for 90 minutes areroom temperature.

[0241] 18. Wash as in Step 14.

[0242] 19. Add 100 μl room temperature ABST/H₂O₂ solution to each well.

[0243] 20. Incubate, with shaking for 15 to 30 minutes at roomtemperature.

[0244] 21. If necessary, stop reaction by adding 100 μl of 0.2 M HCl toeach well.

[0245] 22. Read results on Dynatech MR7000 ELISA reader with test filterat 410 nM and reference filter at 630 nM.

PYK2 BIOASSAY

[0246] This assay is used to measure the in vitro kinase activity of HAepitope-tagged full length pyk2 (FL.pyk2-HA) in an ELISA assay.

[0247] Materials and Reagents

[0248] 1. Coming 96-well ELISA plates.

[0249] 2. 12CA5 monoclonal anti-HA antibody (SUGEN, Inc.)

[0250] 3. PBS (Dulbecco's Phosphate-Buffered Saline (Gibco Catalog #450-1300EB)

[0251] 4. TBST Buffer: for 1 L, mix 8.766 g NaCl, 6.057 g TRIS and 1 mlof 0.1% Triton X-100 in approx. 900 ml dH₂O. Adjust pH to 7.2, bringvolume to 1 L.

[0252] 5. Blocking Buffer: for 1 L, mix 100 g 10% BSA, 12.1 g 100 mMTRIS, 58.44 g 1M NaCl and 10 mL of 1% TWEEN-20.

[0253] 6. FL.pyk2-HA from sf9 cell lysates (SUGEN, Inc.).

[0254] 7. 4% DMSO in MilliQue H₂O.

[0255] 8. 10 mM ATP in dH₂O.

[0256] 9. 1M MnCl₂.

[0257] 10. 1M MgCl₂.

[0258] 11. 1M Dithiothreitol (DTT).

[0259] 12. 10×Kinase buffer phosphorylation: mix 5.0 ml 1M Hepes (pH7.5), 0.2 ml 1M MnCl₂, 1.0 ml 1 M MgCl₂, 1.0 ml 10% Triton X-100 in 2.8ml dH₂O. Just prior to use, add 0.1 ml 1M DTT.

[0260] 13. NUNC 96-well V bottom polypropylene plates.

[0261] 14. 500 mM EDTA in dH₂O.

[0262] 15. Antibody dilution buffer: for 100 mL, 1 mL 5% BSA/PBS and 1mL 10% Tween-20 in 88 mL TBS.

[0263] 16. HRP-conjugated anti-Ptyr (PY99, Santa Cruz Biotech Cat. No.SC-7020).

[0264] 17. ABTS, Moss, Cat. No. ABST-2000.

[0265] 18. 10% SDS.

[0266] Procedure

[0267] 1. Coat Coming 96 well ELISA plates with 0.5 μg per well 12CA5anti-HA antibody in 100 μl PBS. Store overnight at 4° C.

[0268] 2. Remove unbound HA antibody from wells by inverting plate. Washplate with dH₂O. Pat the plate on a paper towel to remove excess liquid.

[0269] 3. Add 150 μl Blocking Buffer to each well. Incubate, withshaking, for 30 min at room temperature.

[0270] 4. Wash plate 4× with TBS-T.

[0271] 5. Dilute lysate in PBS (1.5 μg lysate/100 μl PBS).

[0272] 6. Add 100 μl of diluted lysate to each well. Shake at roomtemperature for 1 hr.

[0273] 7. Wash as in step 4.

[0274] 8. Add 50 μl of 2×kinase Buffer to ELISA plate containingcaptured pyk2-HA.

[0275] 9. Add 25 μL of 400 μM test compound in 4% DMSO to each well. Forcontrol wells use 4% DMSO alone.

[0276] 10. Add 25 μL of 0.5 M EDTA to negative control wells.

[0277] 11. Add 25 μl of 20 μM ATP to all wells. Incubate, with shaking,for 10 minutes.

[0278] 12. Stop reaction by adding 25 μl 500 mM EDTA (pH 8.0) to allwells.

[0279] 13. Wash as in step 4.

[0280] 14. Add 100 μL HRP conjugated anti-Ptyr diluted 1:6000 inAntibody Dilution Buffer to each well. Incubate, with shaking, for 1 hr.at room temperature.

[0281] 15. Wash plate 3× with TBST and 1× with PBS.

[0282] 16. Add 100 μL of ABST solution to each well.

[0283] 17. If necessary, stop the development reaction by adding 20 μL10% SDS to each well.

[0284] 18. Read plate on ELISA reader with test filter at 410 nM andreference filter at 630 nM.

FGFR1 BIOASSAY

[0285] This assay is used to measure the in vitro kinase activity ofFGF1-R in an ELISA assay.

[0286] Materials and Reagents

[0287] 1. Costar 96-well ELISA plates (Coming Catalog # 3369).

[0288] 2. Poly(Glu-Tyr) (Sigma Catalog # P0275).

[0289] 3. PBS (Gibco Catalog # 450-1300EB)

[0290] 4. 50 mM Hepes Buffer Solution.

[0291] 5. Blocking Buffer (5% BSA/PBS).

[0292] 6. Purified GST-FGFR1 (SUGEN, Inc.)

[0293] 7. Kinase Dilution Buffer.

[0294]  Mix 500 μl 1M Hepes (GIBCO), 20 μl 5% BSA/PBS, 10 μl 100 mMsodium orthovanadate and 50 μl 5M NaCl.

[0295] 8. 10 mM ATP

[0296] 9. ATP/MnCl₂ phosphorylation mix: mix 20 μL ATP, 400 μL 1M MnCl₂and 9.56 ml dH₂O.

[0297] 10. NUNC 96well V bottom polypropylene plates (Applied ScientificCatalog # AS-72092).

[0298] 11. 0.5M EDTA.

[0299] 12. 0.05% TBST

[0300]  Add 500 μL TWEEN to 1 liter TBS.

[0301] 13. Rabbit polyclonal anti-phosphotyrosine serum (SUGEN, Inc.).

[0302] 14. Goat anti-rabbit IgG peroxidase conjugate (Biosource, Catalog# ALI0404).

[0303] 15. ABTS Solution. 16. ABTS/H₂O₂ solution.

[0304] Procedure

[0305] 1. Coat Costar 96 well ELISA plates with 1 μg per wellPoly(Glu-Tyr) in 100 μl PBS. Store overnight at 4° C.

[0306] 2. Wash coated plates once with PBS.

[0307] 3. Add 150 μL of 5% BSA/PBS Blocking Buffer to each well.Incubate, with shaking, for 1 hr at room temperature.

[0308] 4. Wash plate 2× with PBS, then once with 50 mM Hepes. Pat plateson a paper towel to remove excess liquid and bubbles.

[0309] 5. Add 25 μL of 0.4 mM test compound in 4% DMSO or 4% DMSO alone(controls) to plate.

[0310] 6. Dilute purified GST-FGFR1 in Kinase Dilution Buffer (5 ngkinase/50 ul KDB/well).

[0311] 7. Add 50 μL of diluted kinase to each well.

[0312] 8. Start kinase reaction by adding 25 μl/well of freshly preparedATP/Mn++ (0.4 ml 1M MnCl₂, 40 μL 10 mM ATP, 9.56 ml dH₂O), freshlyprepared).

[0313] 9. Stop reaction with 25 μL of 0.5M EDTA.

[0314] 10. Wash plate 4× with fresh TBST.

[0315] 11. Make up Antibody Dilution Buffer: For 50 ml, mix 5 ml of 5%BSA, 250 μl of 5% milk and 50 μl of 100 mM sodium vanadate, bring tofinal volume with 0.05% TBST.

[0316] 12. Add 100 μl per well of anti-phosphotyrosine (1:10000 dilutionin ADB). Incubate, with shaking for 1 hr. at room temperature.

[0317] 13. Wash as in step 10.

[0318] 14. Add 100 μl per well of Biosource Goat anti-rabbit IgGperoxidase conjugate (1:6000 dilution in ADB). Incubate, with shakingfor 1 hr. at room temperature.

[0319] 15. Wash as in step 10 and then with PBS to remove bubbles andexcess TWEEN.

[0320] 16. Add 100 μl of ABTS/H₂O₂ solution to each well.

[0321] 17. Incubate, with shaking, for 10 to 20 minutes. Remove anybubbles.

[0322] 18. Read assay on Dynatech MR7000 ELISA reader: test filter at410 nM, reference filter at 630 nM.

EGFR BIOASSAY

[0323] This assay is used to the in vitro kinase activity of EGFR in anELISA assay.

[0324] Materials and Reagents

[0325] 1. Coming 96-well ELISA plates.

[0326] 2. SUMO1 monoclonal anti-EGFR antibody (SUGEN, Inc.).

[0327] 3. PBS.

[0328] 4. TBST Buffer.

[0329] 5. Blocking Buffer: for 100 ml, mix 5.0 g Carnation® InstantNon-fat Milk with 100 ml of PBS.

[0330] 6. A431 cell lysate (SUGEN, Inc.).

[0331] 7. TBS Buffer.

[0332] 8. TBS+10% DMSO: for 1L, mix 1.514 g TRIS, 2.192 g NaCl and 25 mlDMSO; bring to 1 liter total volume with dH₂O.

[0333] 9. ATP (Adenosine-5′-triphosphate, from Equine muscle, Sigma Cat.No. A-5394), 1.0 mM solution in dH₂O. This reagent should be made upimmediately prior to use and kept on ice.

[0334] 10 1.0 mM MnCl₂.

[0335] 11. ATP/MnCl₂ phosphorylation mix: for 10 ml, mix 300 μl of 1 mMATP, 500 μl MnCl₂ and 9.2 ml dH₂O. Prepare just prior to use, keep onice.

[0336] 12. NUNC 96-well V bottom polypropylene plates.

[0337] 13. EDTA.

[0338] 14. Rabbit polyclonal anti-phosphotyrosine serum (SUGEN, Inc.).

[0339] 15. Goat anti-rabbit IgG peroxidase conjugate (Biosource Cat. No.ALI0404).

[0340] 16. ABTS.

[0341] 17. 30% Hydrogen peroxide.

[0342] 18. ABTS/H₂O₂.

[0343] 19. 0.2 M HCl.

[0344] Procedure

[0345] 1. Coat Coming 96 well ELISA plates with 0.5 μg SUMO1 in 100 μlPBS per well, hold overnight at 4° C.

[0346] 2. Remove unbound SUMO1 from wells by inverting plate to removeliquid. Wash 1× with dH₂O. Pat the plate on a paper towel to removeexcess liquid.

[0347] 3. Add 150 μl of Blocking Buffer to each well. Incubate, withshaking, for 30 min. at room temperature.

[0348] 4. Wash plate 3× with deionized water, then once with TBST. Patplate on a paper towel to remove excess liquid and bubbles.

[0349] 5. Dilute lysate in PBS (7 μg lysate/100 μl PBS).

[0350] 6. Add 100 μl of diluted lysate to each well. Shake at roomtemperature for 1 hr.

[0351] 7. Wash plates as in 4, above.

[0352] 8. Add 120 μl TBS to ELISA plate containing captured EGFR.

[0353] 9. Dilute test compound 1:10 in TBS, place in well

[0354] 10. Add 13.5 μl diluted test compound to ELISA plate. To controlwells, add 13.5 μl TBS in 10% DMSO.

[0355] 11. Incubate, with shaking, for 30 minutes at room temperature.

[0356] 12. Add 15 μl phosphorylation mix to all wells except negativecontrol well. Final well volume should be approximately 150 μl with 3 μMATP/5 mM MnCl₂ final concentration in each well. Incubate with shakingfor 5 minutes.

[0357] 13. Stop reaction by adding 16.5 μl of EDTA solution whileshaking. Shake for additional 1 min.

[0358] 14. Wash 4× with deionized water, 2× with TBST.

[0359] 15. Add 100 μl anti-phosphotyrosine (1:3000 dilution in TBST) perwell. Incubate, with shaking, for 30-45 min. at room temperature.

[0360] 16. Wash as in 4, above.

[0361] 17. Add 100 μt Biosource Goat anti-rabbit IgG peroxidaseconjugate (1:2000 dilution in TBST) to each well. Incubate with shakingfor 30 min. at room temperature.

[0362] 18. Wash as in 4, above.

[0363] 19. Add 100 μl of ABTS/H₂O₂ solution to each well.

[0364] 20. Incubate 5 to 10 minutes with shaking. Remove any bubbles.

[0365] 21. If necessary, stop reaction by adding 100 μl 0.2 M HCl perwell.

[0366] 22. Read assay on Dynatech MR7000 ELISA reader: test filter at410 nM, reference filter at 630 nM.

PDGFR BIOASSAY

[0367] This assay is used to the in vitro kinase activity of PDGFR in anELISA assay.

[0368] Materials and Reagents

[0369] 1. Corning 96-well ELISA plates

[0370] 2. 28D4C10 monoclonal anti-PDGFR antibody (SUGEN, Inc.).

[0371] 3. PBS.

[0372] 4. TBST Buffer.

[0373] 5. Blocking Buffer (same as for EGFR bioassay).

[0374] 6. PDGFR-β expressing NIH 3T3 cell lysate (SUGEN, Inc.).

[0375] 7. TBS Buffer.

[0376] 8. TBS+10% DMSO.

[0377] 9. ATP.

[0378] 10. MnCl₂.

[0379] 11. Kinase buffer phosphorylation mix: for 10 ml, mix 250 μl 1MTRIS, 200 μl 5M NaCl, 100 μl 1M MnCl₂ and 50 μl 100 mM Triton X-100 inenough dH₂O to make 10 ml.

[0380] 12. NUNC 96-well V bottom polypropylene plates.

[0381] 13. EDTA.

[0382] 14. Rabbit polyclonal anti-phosphotyrosine serum (SUGEN, Inc.).

[0383] 15. Goat anti-rabbit IgG peroxidase conjugate (Biosource Cat. No.ALI0404).

[0384] 16. ABTS.

[0385] 17. Hydrogen peroxide, 30% solution.

[0386] 18. ABTS/H₂O₂.

[0387] 19. 0.2 M HCl.

[0388] Procedure

[0389] 1. Coat Corning 96 well ELISA plates with 0.5 μg 28D4C10 in 100μl PBS per well, hold overnight at 4° C.

[0390] 2. Remove unbound 28D4C10 from wells by inverting plate to removeliquid. Wash 1× with dH₂O. Pat the plate on a paper towel to removeexcess liquid.

[0391] 3. Add 150 μl of Blocking Buffer to each well. Incubate for 30min. at room temperature with shaking.

[0392] 4. Wash plate 3× with deionized water, then once with TBST. Patplate on a paper towel to remove excess liquid and bubbles.

[0393] 5. Dilute lysate in HNTG (10 μg lysate/100 μl HNTG).

[0394] 6. Add 100 μl of diluted lysate to each well. Shake at roomtemperature for 60 min.

[0395] 7. Wash plates as described in Step 4.

[0396] 8. Add 80 μl working kinase buffer mix to ELISA plate containingcaptured PDGFR.

[0397] 9. Dilute test compound 1:10 in TBS in 96-well polypropyleneplates.

[0398] 10. Add 10 μl diluted test compound to ELISA plate. To controlwells, add 10 μl TBS+10% DMSO. Incubate with shaking for 30 minutes atroom temperature.

[0399] 11. Add 10 μl ATP directly to all wells except negative controlwell (final well volume should be approximately 100 μl with 20 μM ATP ineach well.) Incubate 30 minutes with shaking.

[0400] 12. Stop reaction by adding 10 μl of EDTA solution to each well.

[0401] 13. Wash 4× with deionized water, twice with TBST.

[0402] 14. Add 100 μl anti-phosphotyrosine (1:3000 dilution in TBST) perwell. Incubate with shaking for 30-45 min. at room temperature.

[0403] 15. Wash as in Step 4.

[0404] 16. Add 100 μl Biosource Goat anti-rabbit IgG peroxidaseconjugate (1:2000 dilution in TBST) to each well. Incubate with shakingfor 30 min. at room temperature.

[0405] 17. Wash as in Step 4.

[0406] 18. Add 100 μl of ABTS/H₂O₂ solution to each well.

[0407] 19. Incubate 10 to 30 minutes with shaking. Remove any bubbles.

[0408] 20. If necessary stop reaction with the addition of 100 μl 0.2 MHCl per well.

[0409] 21. Read assay on Dynatech MR7000 ELISA reader with test filterat 410 nM and reference filter at 630 nM.

CELLULAR HER-2 KINASE ASSAY

[0410] This assay is used to measure HER-2 kinase activity in wholecells in an ELISA format.

[0411] Materials and Reagents

[0412] 1. DMEM (GIBCO Catalog # 11965-092).

[0413] 2. Fetal Bovine Serum (FBS, GIBCO Catalog # 16000-044), heatinactivated in a water bath for 30 min. at 56° C.

[0414] 3. Trypsin (GIBCO Catalog # 25200-056).

[0415] 4. L-Glutamine (GIBCO Catalog # 25030-081).

[0416] 5. HEPES (GIBCO Catalog # 15630-080).

[0417] 6. Growth Media: Mix 500 ml DMEM, 55 ml heat inactivated FBS, 10ml HEPES and 5.5 ml L-Glutamine.

[0418] 7. Starve Media: Mix 500 ml DMEM, 2.5 ml heat inactivated FBS, 10ml HEPES and 5.5 ml L-Glutamine.

[0419] 8. PBS.

[0420] 9. Flat Bottom 96-well Tissue Culture Micro Titer Plates (ComingCatalog # 25860).

[0421] 10. 15 cm Tissue Culture Dishes (Coming Catalog # 08757148).

[0422] 11. Coming 96-well ELISA Plates.

[0423] 12. NUNC 96-well V bottom polypropylene plates.

[0424] 13. Costar Transfer Cartridges for the Transtar 96 (CostarCatalog # 7610).

[0425] 14. SUMO 1: monoclonal anti-EGFR antibody (SUGEN, Inc.).

[0426] 15. TBST Buffer.

[0427] 16. Blocking Buffer: 5% Carnation Instant Milk® in PBS.

[0428] 17. EGF Ligand: EGF-201, Shinko American, Japan. Suspend powderin 100 μL of 10 mM HCl. Add 100 μL 10 mM NaOH. Add 800 μL PBS andtransfer to an Eppendorf tube, store at −20° C. until ready to use.

[0429] 18. HNTG Lysis Buffer: For Stock 5×HNTG, mix 23.83 g Hepes, 43.83g NaCl, 500 ml glycerol and 100 ml Triton X-100 and enough dH₂O to make1 L of total solution.

[0430]  For 1×HNTG*, mix 2 ml 5×HNTG, 100 μL 0.1M Na₃VO₄, 250 μL 0.2MNa₄P₂O₇ and 100 μL EDTA.

[0431] 19. EDTA.

[0432] 20. Na₃VO₄: To make stock solution, mix 1.84 g Na₃VO₄ with 90 mldH₂O. Adjust pH to 10. Boil in microwave for one minute (solutionbecomes clear). Cool to room temperature. Adjust pH to 10. Repeatheating/cooling cycle until pH remains at 10.

[0433] 21. 200 mM Na₄P₂O₇.

[0434] 22. Rabbit polyclonal antiserum specific for phosphotyrosine(anti-Ptyr antibody, SUGEN, Inc.).

[0435] 23. Affinity purified antiserum, goat anti-rabbit IgG antibody,peroxidase conjugate (Biosource Cat # ALI0404).

[0436] 24. ABTS Solution.

[0437] 25. 30% Hydrogen peroxide solution.

[0438] 26. ABTS/H₂O₂.

[0439] 27. 0.2 M HCl.

[0440] Procedure

[0441] 1. Coat Corning 96 well ELISA plates with SUMO1 at 1.0 μg perwell in PBS, 100 μl final volume/well. Store overnight at 4° C.

[0442] 2. On day of use, remove coating buffer and wash plate 3 timeswith dH₂O and once with TBST buffer. All washes in this assay should bedone in this manner, unless otherwise specified.

[0443] 3. Add 100 μL of Blocking Buffer to each well. Incubate plate,with shaking, for 30 min. at room temperature. Just prior to use, washplate.

[0444] 4. Use EGFr/HER-2 chimera/3T3-C7 cell line for this assay.

[0445] 5. Choose dishes having 80-90% confluence. Collect cells bytrypsinization and centrifuge at 1000 rpm at room temperature for 5 min.

[0446] 6. Resuspend cells in starve medium and count with trypan blue.Viability above 90% is required. Seed cells in starve medium at adensity of 2,500 cells per well, 90 μL per well, in a 96 well microtiterplate. Incubate seeded cells overnight at 37° under 5% CO₂.

[0447] 7. Start the assay two days after seeding.

[0448] 8. Test compounds are dissolved in 4% DMSO. Samples are thenfurther diluted directly on plates with starve-DMEM. Typically, thisdilution will be 1:10 or greater. All wells are then transferred to thecell plate at a further 1:10 dilution (10 μl sample and media into 90 μlof starve media). The final DMSO concentration should be 1% or lower. Astandard serial dilution may also be used.

[0449] 9. Incubate under 5% CO₂ at 37° C. for 2 hours.

[0450] 10. Prepare EGF ligand by diluting stock EGF (16.5 μM) in warmDMEM to 150 nM.

[0451] 11. Prepare fresh HNTG* sufficient for 100 μL per well; place onice.

[0452] 12. After 2 hour incubation with test compound, add prepared EGFligand to cells, 50 μL per well, for a final concentration of 50 nM.Positive control wells receive the same amount of EGF. Negative controlsdo not receive EGF. Incubate at 37° C. for 10 min.

[0453] 13. Remove test compound, EGF, and DMEM. Wash cells once withPBS.

[0454] 14. Transfer HNTG* to cells, 100 μL per well. Place on ice for 5minutes. Meanwhile, remove blocking buffer from ELISA plate and wash.

[0455] 15. Scrape cells from plate with a micropipettor and homogenizecell material by repeatedly aspirating and dispensing the HNTG* lysisbuffer. Transfer lysate to a coated, blocked, washed ELISA plate.

[0456] 16. Incubate, with shaking, at room temperature for 1 hr.

[0457] 17. Remove lysate, wash. Transfer freshly diluted anti-Ptyrantibody (1:3000 in TBST) to ELISA plate, 100 μL per well.

[0458] 18. Incubate, with shaking, at room temperature, for 30 min.

[0459] 19. Remove anti-Ptyr antibody, wash. Transfer freshly dilutedBIOSOURCE antibody to ELISA plate(1 :8000 in TBST, 100 μL per well).

[0460] 20. Incubate, with shaking, at room temperature for 30 min.

[0461] 21. Remove BIOSOURCE antibody, wash. Transfer freshly preparedABTS/H₂O₂ solution to ELISA plate, 100 μL per well.

[0462] 22. Incubate, with shaking, for 5-10 minutes. Remove any bubbles.

[0463] 23. Stop reaction by adding 100 μL of 0.2M HCl per well.

[0464] 24. Read assay on Dynatech MR7000 ELISA reader with test filterset at 410 nM and reference filter at 630 nM.

CDK2/CYCLIN A ASSAY

[0465] This assay is used to measure the in vitro serine/threoninekinase activity of human cdk2/cyclin A in a Scintillation ProximityAssay (SPA).

[0466] Materials and Reagents

[0467] 1. Wallac 96-well polyethylene terephthalate (flexi) plates(Wallac Catalog # 1450-401).

[0468] 2. Amersham Redivue [γ³³P] ATP (Amersham catalog # AH 9968).

[0469] 3. Amersham streptavidin coated polyvinyltoluene SPA beads(Amersham catalog # RPNQ0007). The beads should be reconstituted in PBSwithout magnesium or calcium, at 20 mg/ml.

[0470] 4. Activated cdk2/cyclin A enzyme complex purified from Sf9 cells(SUGEN, Inc.).

[0471] 5. Biotinylated peptide substrate (Debtide). Peptidebiotin-X-PKTPKKAKKL is dissolved in dH₂O at a concentration of 5 mg/ml.

[0472] 6. 20% DMSO in dH₂O.

[0473] 7. Kinase buffer: for 10 ml, mix 9.1 ml dH₂O, 0.5 ml TRIS(pH7.4), 0.2 ml 1M MgCl₂, 0.2 ml 10% NP40 and 0.02 ml 1M DTT, added freshjust prior to use.

[0474] 8. 10 mM ATP in dH₂O.

[0475] 9. 1M Tris, pH adjusted to 7.4 with HCl.

[0476] 10. 1M MgCl₂.

[0477] 11. 1M DTT.

[0478] 12. PBS (Gibco Catalog # 14190-144).

[0479] 13. 0.5M EDTA.

[0480] 14. Stop solution: For 10 ml, mix 9.25 ml PBS, 0.05 ml 10 mM ATP,0.1 ml 0.5 M EDTA, 0.1 ml 10% Triton X-100 and 1.5 ml of 50 mg/ml SPAbeads.

[0481] Procedure

[0482] 1. Prepare solutions of test compounds at 4× the desired finalconcentration in 5% DMSO. Add 10 μL to each well. For positive andnegative controls, use 10 μL 20% DMSO alone in wells.

[0483] 2. Dilute the peptide substrate (deb-tide) 1:250 with dH₂O togive a final concentration of 0.02 mg/ml.

[0484] 3. Mix 24 μL 0.1 mM ATP with 24 μCi γ³³P ATP and enough dH₂O tomake 600 μL.

[0485] 4. Mix diluted peptide and ATP solutions 1:1 (600 μL+600 μL perplate). Add 10 μL of this solution to each well.

[0486] 5. Dilute 5 μL of cdk2/cyclin A solution into 2.1 ml 2×kinasebuffer (per plate). Add 20 μL enzyme per well. For negative controls,add 20 μL 2×kinase buffer without enzyme.

[0487] 6. Mix briefly on a plate shaker; incubate for 60 minutes.

[0488] 7. Add 200 μL stop solution per well.

[0489] 8. Let stand at least 10 min.

[0490] 9. Spin plate at approx. 2300 rpm for 10-15 min.

[0491] 10. Count plate on Trilux reader.

MET TRANSPHOSPHORYLATION ASSAY

[0492] This assay is used to measure phosphotyrosine levels on apoly(glutamic acid:tyrosine, 4:1) substrate as a means for identifyingagonists/antagonists of met transphosphorylation of the substrate.

[0493] Materials and Reagents

[0494] 1. Corning 96-well ELISA plates, Corning Catalog # 25805-96.

[0495] 2. Poly(glu-tyr), 4:1, Sigma, Cat. No; P 0275.

[0496] 3. PBS, Gibco Catalog # 450-1300EB

[0497] 4. 50 mM HEPES

[0498] 5. Blocking Buffer: Dissolve 25 g Bovine Serum Albumin, SigmaCat. No A-7888, in 500 ml PBS, filter through a 4 μm filter.

[0499] 6. Purified GST fusion protein containing the Met kinase domain,SUGEN, Inc.

[0500] 7. TBST Buffer.

[0501] 8. 10% aqueous (MilliQue H₂O) DMSO.

[0502] 9. 10 mM aqueous (dH₂O) Adenosine-5′-triphosphate, Sigma Cat. No.A-5394.

[0503] 10. 2×Kinase Dilution Buffer: for 100 ml, mix 10 mL 1M HEPES atpH 7.5 with 0.4 mL 5% BSA/PBS, 0.2 mL 0.1 M sodium orthovanadate and 1mL 5M sodium chloride in 88.4 mL dH₂O.

[0504] 11. 4×ATP Reaction Mixture: for 10 mL, mix 0.4 mL 1 M manganesechloride and 0.02 mL 0.1 M ATP in 9.56 mL dH₂O.

[0505]12. 4×Negative Controls Mixture: for 10 mL, mix 0.4 mL 1 Mmanganese chloride in 9.6 mL dH₂O.

[0506] 13. NUNC 96-well V bottom polypropylene plates, AppliedScientific Catalog # S-72092

[0507]14. 500 mM EDTA.

[0508] 15. Antibody Dilution Buffer: for 100 mL, mix 10 mL 5% BSA/PBS,0.5 mL 5% Carnation® Instant Milk in PBS and 0.1 mL 0.1 M sodiumorthovanadate in 88.4 mL TBST.

[0509] 16. Rabbit polyclonal antophosphotyrosine antibody, SUGEN, Inc.

[0510] 17. Goat anti-rabbit horseradish peroxidase conjugated antibody,Biosource, Inc.

[0511] 18. ABTS Solution: for 1 L, mix 19.21 g citric acid, 35.49 gNA₂HPO₄ and 500 mg ABTS with sufficient dH₂O to make 1 L.

[0512] 19. ABTS/H₂O₂: mix 15 mL ABST solution with 2 μL H₂O₂ fiveminutes before use.

[0513] 20. 0.2 M HCl

[0514] Procedure

[0515] 1. Coat ELISA plates with 2 μg Poly(Glu-Tyr) in 100 μL PBS, holdovernight at 4° C.

[0516] 2. Block plate with 150 μL of 5% BSA/PBS for 60 min.

[0517] 3. Wash plate twice with PBS then once with 50 mM Hepes buffer pH7.4.

[0518] 4. Add 50 μl of the diluted kinase to all wells. (Purified kinaseis diluted with Kinase Dilution Buffer. Final concentration should be 10ng/well.)

[0519] 5. Add 25 μL of the test compound (in 4% DMSO) or DMSO alone (4%in dH₂O) for controls to plate.

[0520] 6. Incubate the kinase/compound mixture for 15 minutes.

[0521] 7. Add 25 μL of 40 mM MnCl₂ to the negative control wells.

[0522] 8. Add 25 μL ATP/ MnCl₂ mixture to the all other wells (exceptthe negative controls). Incubate for 5 min.

[0523] 9. Add 25 μL 500 mM EDTA to stop reaction.

[0524] 10. Wash plate 3× with TBST.

[0525] 11. Add 100 μL rabbit polyclonal anti-Ptyr diluted 1:10,000 inAntibody Dilution Buffer to each well. Incubate, with shaking, at roomtemperature for one hour.

[0526] 12. Wash plate 3× with TBST.

[0527] 13. Dilute Biosource HRP conjugated anti-rabbit antibody 1:6,000in Antibody Dilution buffer. Add 100 μL per well and incubate at roomtemperature, with shaking, for one hour.

[0528] 14. Wash plate 1× X with PBS.

[0529] 15. Add 100 μl of ABTS/H₂O₂ solution to each well.

[0530] 16. If necessary, stop the development reaction with the additionof 100 μl of 0.2M HCl per well.

[0531] 17. Read plate on Dynatech MR7000 ELISA reader with the testfilter at 410 nM and the reference filter at 630 nM.

IGF-1 Transphosphorylation Assay

[0532] This assay is used to measure the phosphotyrosine level inpoly(glutamic acid:tyrosine, 4:1) for the identification ofagonists/antagonists of gst-IGF-1 transphosphorylation of a substrate.

[0533] Materials and Reagents

[0534] 1. Corning 96-well ELISA plates.

[0535] 2. Poly(Glu-Tyr),4:1, Sigma Cat. No. P 0275.

[0536] 3. PBS, Gibco Catalog # 450-1300EB.

[0537] 4. 50 mM HEPES

[0538] 5. TBB Blocking Buffer: for 1 L, mix 100 g BSA, 12.1 g TRIS (pH7.5), 58.44 g sodium chloride and 10 mL 1% TWEEN-20.

[0539] 6. Purified GST fusion protein containing the IGF-1 kinase domain(SUGEN, Inc.)

[0540] 7. TBST Buffer: for 1 L, mix 6.057 g Tris, 8.766 g sodiumchloride and 0.5 ml TWEEN-20 with enough dH₂O to make 1 liter.

[0541] 8. 4% DMSO in Milli-Q H₂O.

[0542]9. 10 mM ATP in dH₂O.

[0543] 10. 2×Kinase Dilution Buffer: for 100 mL, mix 10 mL 1 M HEPES (pH7.5), 0.4 mL 5% BSA in dH₂O, 0.2 mL 0.1 M sodium orthovanadate and 1 mL5 M sodium chloride with enough dH₂O to make 100 mL.

[0544] 11. 4×ATP Reaction Mixture: for 10 mL, mix 0.4 mL 1 M MnCl₂ and0.008 mL 0.01 M ATP and 9.56 mL dH₂O.

[0545] 12. 4×Negative Controls Mixture: mix 0.4 mL 1 M MnCl₂ in 9.60 mLdH₂O.

[0546] 13. NUNC 96-well V bottom polypropylene plates.

[0547] 14. 500 mM EDTA in dH₂O.

[0548] 15. Antibody Dilution Buffer: for 100 mL, mix 10 mL 5% BSA inPBS, 0.5 mL 5% Carnation Instant Non-fat Milk in PBS and 0.1 mL 0.1 Msodium orthovanadate in 88.4 mL TBST.

[0549] 16. Rabbit Polyclonal antiphosphotyrosine antibody, SUGEN, Inc.

[0550] 17. Goat anti-rabbit HRP conjugated antibody, Biosource.

[0551] 18. ABTS Solution.

[0552] 20. ABTS/H₂O₂: mix 15 mL ABTS with 2 μL H₂O₂ 5 minutes beforeusing.

[0553] 21. 0.2 M HCl in dH₂O.

[0554] Procedure

[0555] 1. Coat ELISA plate with 2.0 μg/well Poly(Glu, Tyr), 4:1 (SigmaP0275) in 100 μl PBS. Store plate overnight at 4° C.

[0556] 2. Wash plate once with PBS.

[0557] 3. Add 100 μl of TBB Blocking Buffer to each well. Incubate platefor 1 hour with shaking at room temperature.

[0558] 4. Wash plate once with PBS, then twice with 50 mM Hepes bufferpH 7.5.

[0559] 5. Add 25 μL of test compound in 4% DMSO (obtained by diluting astock solution of 10 mM test compound in 100% DMSO with dH₂O) to plate.

[0560] 6. Add 10.0 ng of gst-IGF-1 kinase in 50 μl Kinase DilutionBuffer to all wells.

[0561] 7. Start kinase reaction by adding 25 μl 4×ATP Reaction Mixtureto all test wells and positive control wells. Add 25 μl 4×NegativeControls Mixture to all negative control wells. Incubates for 10minutes, with shaking, at room temperature.

[0562] 8. Add 25 μl 0.5M EDTA (pH 8.0) to all wells.

[0563] 9. Wash plate 4× with TBST Buffer.

[0564] 10. Add rabbit polyclonal anti-phosphotyrosine antisera at adilution of 1:10,000 in 100 μl Antibody Dilution Buffer to all wells.Incubate, with shaking, at room temperature for 1 hour.

[0565] 11. Wash plate as in step 9.

[0566] 12. Add 100 μL Biosource anti-rabbit HRP at a dilution of1:10,000 in Antibody dilution buffer to all wells. Incubate, withshaking, at room temperature for 1 hour.

[0567] 13. Wash plate as in step 9, follow with one wash with PBS toremove bubbles and excess Tween-20.

[0568] 14. Develop by adding 100μl/well ABTS/H₂O₂ to each well

[0569] 15. After about 5 minutes, read on ELISA reader with test filterat 410 nm and referenced filter at 630 nm.

BrdU INCORPORATION ASSAYS

[0570] The following assays use cells engineered to express a selectedreceptor and then evaluate the effect of a compound of interest on theactivity of ligand-induced DNA synthesis by determining BrdUincorporation into the DNA.

[0571] The following materials, reagents and procedure are general toeach of the following BrdU incorporation assays. Variances in specificassays are noted.

[0572] General Materials and Reagents

[0573] 1. The appropriate ligand.

[0574] 2. The appropriate engineered cells.

[0575] 3. BrdU Labeling Reagent: 10 mM, in PBS, pH7.4 (Roche MolecularBiochemicals, Indianapolis, Ind.).

[0576] 4. FixDenat: fixation solution (Roche Molecular Biochemicals,Indianapolis, Ind.).

[0577] 5. Anti-BrdU-POD: mouse monoclonal antibody conjugated withperoxidase (Chemicon, Temecula, Calif.).

[0578] 6. TMB Substrate Solution: tetramethylbenzidine (TMB, ready touse, Roche Molecular Biochemicals, Indianapolis, Ind.).

[0579] 7. PBS Washing Solution: 1×PBS, pH 7.4.

[0580] 8. Albumin, Bovine (BSA), fraction V powder (Sigma Chemical Co.,USA).

[0581] General Procedure

[0582] 1. Cells are seeded at 8000 cells/well in 10% CS, 2 mM Gln inDMEM, in a 96 well plate. Cells are incubated overnight at 37° C. in 5%CO₂.

[0583] 2. After 24 hours, the cells are washed with PBS, and then areserum-starved in serum free medium (0% CS DMEM with 0.1% BSA) for 24hours.

[0584] 3. On day 3, the appropriate ligand and the test compound areadded to the cells simultaneously. The negative control wells receiveserum free DMEM with 0.1% BSA only; the positive control cells receivethe ligand but no test compound. Test compounds are prepared in serumfree DMEM with ligand in a 96 well plate, and serially diluted for 7test concentrations.

[0585] 4. After 18 hours of ligand activation, diluted BrdU labelingreagent (1:100 in DMEM, 0.1% BSA) is added and the cells are incubatedwith BrdU (final concentration is 10 μM) for 1.5 hours.

[0586] 5. After incubation with labeling reagent, the medium is removedby decanting and tapping the inverted plate on a paper towel. FixDenatsolution is added (50 μl/well) and the plates are incubated at roomtemperature for 45 minutes on a plate shaker.

[0587] 6. The FixDenat solution is removed by decanting and tapping theinverted plate on a paper towel. Milk is added (5% dehydrated milk inPBS, 200 μl/well) as a blocking solution and the plate is incubated for30 minutes at room temperature on a plate shaker.

[0588] 7. The blocking solution is removed by decanting and the wellsare washed once with PBS. Anti-BrdU-POD solution is added (1:200dilution in PBS, 1% BSA, 50 μl/well) and the plate is incubated for 90minutes at room temperature on a plate shaker.

[0589] 8. The antibody conjugate is removed by decanting and rinsing thewells 5 times with PBS, and the plate is dried by inverting and tappingon a paper towel.

[0590] 9. TMB substrate solution is added (100 μl/well) and incubatedfor 20 minutes at room temperature on a plate shaker until colordevelopment is sufficient for photometric detection.

[0591] 10. The absorbance of the samples are measured at 410 nm (in“dual wavelength” mode with a filter reading at 490 nm, as a referencewavelength) on a Dynatech ELISA plate reader.

EGF-Induced BrdU Incorporation Assay

[0592] Materials and Reagents

[0593] 1. Mouse EGF, 201 (Toyobo Co., Ltd., Japan).

[0594] 2. 3T3/EGFRc7.

[0595] Remaining Materials and Reagents and Procedure, as above.

EGF-Induced Her-2-driven BrdU Incorporation Assay

[0596] Materials and Reagents

[0597] 1. Mouse EGF, 201 (Toyobo Co., Ltd., Japan).

[0598] 2. 3T3/EGFr/Her2/EGFr (EGFr with a Her-2 kinase domain).

[0599] Remaining Materials and Reagents and Procedure, as above.

EGF-Induced Her-4-driven BrdU Incorporation Assay

[0600] Materials and Reagents

[0601] 1. Mouse EGF, 201 (Toyobo Co., Ltd., Japan).

[0602] 2. 3T3/EGFr/Her4/EGFr (EGFr with a Her-4 kinase domain).

[0603] Remaining Materials and Reagents and Procedure, as above.

PDGF-Induced BrdU Incorporation Assay

[0604] Materials and Reagents

[0605] 1. Human PDGF B/B (Boehringer Mannheim, Germany).

[0606] 2. 3T3/EGFRc7.

[0607] Remaining Materials and Reagents and Procedure, as above.

FGF-Induced BrdU Incorporation Assay

[0608] Materials and Reagents

[0609] 1. Human FGF2/bFGF (Gibco BRL, USA).

[0610] 2. 3T3c7/EGFr

[0611] Remaining Materials and Reagents and Procedure, as above.

IGF1-Induced BrdU Incorporation Assay

[0612] Materials and Reagents

[0613] 1. Human, recombinant (G511, Promega Corp., USA)

[0614] 2. 3T3/IGF1r.

[0615] Remaining Materials and Reagents and Procedure, as above.

Insulin-Induced BrdU Incorporation Assay

[0616] Materials and Reagents

[0617] 1. Insulin, crystalline, bovine, Zinc (13007, Gibco BRL, USA).

[0618] 2. 3T3/H25.

[0619] Remaining Materials and Reagents and Procedure, as above.

HGF-Induced BrdU Incorporation Assay

[0620] Materials and Reagents

[0621] 1. Recombinant human HGF (Cat. No. 249-HG, R&D Systems, Inc.USA).

[0622] 2. BxPC-3 cells (ATCC CRL-1687).

[0623] Remaining Materials and Reagents, as above.

[0624] Procedure

[0625] 1. Cells are seeded at 9000 cells/well in RPMI 10% FBS in a 96well plate. Cells are incubated overnight at 37° C. in 5% CO₂.

[0626] 2. After 24 hours, the cells are washed with PBS, and then areserum starved in 100 μl serum-free medium (RPMI with 0.1% BSA) for 24hours.

[0627] 3. On day 3, 25 μl containing ligand (prepared at 1 μg/ml in RPMIwith 0.1% BSA; final HGF conc. is 200 ng/ml) and test compounds areadded to the cells. The negative control wells receive 25 μl serum-freeRPMI with 0.1% BSA only; the positive control cells receive the ligand(HGF) but no test compound. Test compounds are prepared at 5 times theirfinal concentration in serum-free RPMI with ligand in a 96 well plate,and serially diluted to give 7 test concentrations. Typically, thehighest final concentration of test compound is 100 μM, and 1:3dilutions are used (i.e. final test compound concentration range is0.137-100 μM).

[0628] 4. After 18 hours of ligand activation, 12.5 μl of diluted BrdUlabeling reagent (1:100 in RPMI, 0.1% BSA) is added to each well and thecells are incubated with BrdU (final concentration is 10 μM) for 1 hour.

[0629] 5. Same as General Procedure.

[0630] 6. Same as General Procedure.

[0631] 7. The blocking solution is removed by decanting and the wellsare washed once with PBS. Anti-BrdU-POD solution (1:100 dilution in PBS,1% BSA) is added (100 μl/well) and the plate is incubated for 90 minutesat room temperature on a plate shaker.

[0632] 8. Same as General Procedure.

[0633] 9. Same as General Procedure.

[0634] 10. Same as General Procedure.

Exponential BrdU Incorporation Assay

[0635] This assay is used to measure the proliferation (DNA synthesis)of exponentially growing A431 cells. The assay will screen for compoundsthat inhibit cell cycle progression.

[0636] Materials and Reagents

[0637] Healthy growing A431 cells. The remainder of the Materials andReagents are the same as listed above in the general protocol section.

[0638] Procedure

[0639] 1. A431 cells are seeded at 8000 cells/well in 10% FBS, 2 mM Glnin DMEM, on a 96-well plate. Cells are incubated overnight at 37° C. in5% CO₂.

[0640] 2. On day 2, test compounds are serially diluted to 7 testconcentrations in the same growth medium on a 96-well plate and then areadded to the cells on a 96-well tissue culture plate.

[0641] 3. After 20-24 hours of incubation, diluted BrdU labeling reagent(1:100 in DMEM, 0.1% BSA) is added and the cells are incubated with BrdU(final concentration is 10 μM) for 2 hours.

[0642] Steps 5-10 of the General Procedure are used to complete theassay.

ZenSrc Assay

[0643] This assay is used to screen for inhibitors of the tyrosinekinase Src.

[0644] Materials and Reagents

[0645] 1. Coating buffer: PBS containing sodium azide (0.2 mg/ml).

[0646] 2. 1% w/vBSA in PBS.

[0647] 3. Wash buffer: PBS containing 0.05% v/v Tween 20 (PBS-TWEEN)

[0648] 4. 500 mM HEPES pH7.4.

[0649] 5. ATP (40 μM)+MgCl₂ (80 mM) in distilled water.

[0650] 6. MgCl₂ (80 mM) in distilled water (for no ATP blanks).

[0651] 7. Test compounds, 10 mM in DMSO.

[0652] 8. Assay Buffer: 100 mM HEPES, pH 7.4, containing 2 mM DTT, 0.2mM sodium orthovanadate and 0.2 mgs/ml BSA.

[0653] 9. Partially purified recombinant human Src (UBI (14-117)

[0654] 10. Anti-phosphotyrosine (SUGEN rabbit polyclonal anti-PY).

[0655] 11. HRP-linked goat anti-rabbit Ig (Biosource International #6430)

[0656] 12. HRP substrate ABTS or Pierce Peroxidase substrate.

[0657] 13. Corning ELISA plates.

[0658] Procedure

[0659] 1. Coat plates with 100 μl of 20 μg/ml poly(Glu-Tyr) (Sigma Cat.No.P0275) containing 0.01% sodium azide. Hold overnight at 4° C.

[0660] 2. Block with 1% BSA at 100 μl/well for one hour at roomtemperature.

[0661] 3. Plate test compounds (10 mM in DMSO) at 2 ul/well on a Costarplate ready for dilution with dH₂O and plating to reaction plates.

[0662] 4. Dilute Src kinase 1:10,000 in Reaction Buffer, for 5 platesprepare 25 ml as follows: 2.5 mls 1M HEPES pH7.4 (stored sterile at 4°C.), 21.85 ml distilled water, 0.1 ml 5% BSA, 0.5 ml 10 mM sodiumorthovanadate (stored sterile at 4° C.), 50 μl 1.0M DTT (stored frozenat −20° C.), and 2.5 μl Src Kinase (stored frozen at −80° C.).

[0663] 5. Add 48 μl of distilled water to the 2 μl of each compound inthe dilution plate then add 25 μl/well of this to the reaction plate.

[0664] 6. Add 50 μl of HRP to each reaction buffer well and then 25 μlATP-MgCl₂/well (MgCl₂ only to no ATP blanks). Incubate at roomtemperature for 15 minutes on plate shaker. Stop reaction by adding 25μl of 0.5M EDTA to each well.

[0665] 7. Wash 4× with PBS-TWEEN.

[0666] 8. Add 100 μl anti-phosphotyrosine (1:10,000 of anti-pTyr serumor 1:3,000 of 10% glycerol diluted PA-affinity purified antibody) inPBS-TWEEN containing 0.5% BSA, 0.025% Non-fat milk powder and 100 μMsodium orthovanadate. Incubate with continuous shaking at roomtemperature for one hour.

[0667] 9. Wash plates 4× with PBS-TWEEN.

[0668] 10. Add 100 μl HRP-linked Ig (1:5,000) in PBS-TWEEN containing0.5% BSA, 0.025% Non-fat milk powder, 100 μM sodium orthovanadate.Incubate with shaking at room temperature for one hour.

[0669] 11. Wash plates 4× with PBS-TWEEN and then once with PBS.

[0670] 12. Develop plate using ABTS or other peroxidase substrate.

Cell Cycle Analysis

[0671] A431 cells in standard growth medium are exposed to a desiredconcentration of a test compound for 20-24 hours at 37° C. The cells arethen collected, suspended in PBS, fixed with 70% ice-cold methanol andstained with propidium iodide. The DNA content is then measured using aFACScan flow cytometer. Cell cycle phase distribution can then beestimated using CeliFIT software (Becton-Dickinson).

HUV-EC-C Assay

[0672] This assay is used to measure a compound's activity againstPDGF-R, FGF-R, VEGF, aFGF or Flk-1/KDR, all of which are naturallyexpressed by HUV-EC cells.

[0673] DAY 0

[0674] 1. Wash and trypsinize HUV-EC-C cells (human umbilical veinendothelial cells, (American Type Culture Collection, catalogue no. 1730CRL). Wash with Dulbecco's phosphate-buffered saline (D-PBS, obtainedfrom Gibco BRL, catalogue no. 14190-029) 2 times at about 1 ml/10 cm² oftissue culture flask. Trypsinize with 0.05% trypsin-EDTA innon-enzymatic cell dissociation solution (Sigma Chemical Company,catalogue no. C-1544). The 0.05% trypsin is made by diluting 0.25%trypsin/1 mM EDTA (Gibco, catalogue no. 25200-049) in the celldissociation solution. Trypsinize with about 1 ml/25-30 cm² of tissueculture flask for about 5 minutes at 37° C. After cells have detachedfrom the flask, add an equal volume of assay medium and transfer to a 50ml sterile centrifuge tube (Fisher Scientific, catalogue no. 05-539-6).

[0675] 2. Wash the cells with about 35 ml assay medium in the 50 mlsterile centrifuge tube by adding the assay medium, centrifuge for 10minutes at approximately 200×g, aspirate the supernatant, and resuspendwith 35 ml D-PBS. Repeat the wash two more times with D-PBS, resuspendthe cells in about 1 ml assay medium/15 cm² of tissue culture flask.Assay medium consists of F12K medium (Gibco BRL, catalogue no.21127-014) and 0.5% heat-inactivated fetal bovine serum. Count the cellswith a Coulter Counter® (Coulter Electronics, Inc.) and add assay mediumto the cells to obtain a concentration of 0.8-1.0×10⁵ cells/ml.

[0676] 3. Add cells to 96-well flat-bottom plates at 100 μl/well or0.8-1.0×10⁴ cells/well, incubate ˜24 h at 37° C., 5% CO₂.

[0677] DAY 1

[0678] 1. Make up two-fold test compound titrations in separate 96-wellplates, generally 50 μM on down to 0 μM. Use the same assay medium asmentioned in day 0, step 2 above. Titrations are made by adding 90μl/well of test compound at 200 μM (4× the final well concentration) tothe top well of a particular plate column. Since the stock test compoundis usually 20 mM in DMSO, the 200 μM drug concentration contains 2%DMSO.

[0679] A diluent made up to 2% DMSO in assay medium (F12K+0.5% fetalbovine serum) is used as diluent for the test compound titrations inorder to dilute the test compound but keep the DMSO concentrationconstant. Add this diluent to the remaining wells in the column at 60μl/well. Take 60 μl from the 120 μl of 200 μM test compound dilution inthe top well of the column and mix with the 60 μl in the second well ofthe column. Take 60 μl from this well and mix with the 60 μl in thethird well of the column, and so on until two-fold titrations arecompleted. When the next-to-the-last well is mixed, take 60 μl of the120 μl in this well and discard it. Leave the last well with 60 μl ofDMSO/media diluent as a non-test compound-containing control. Make 9columns of titrated test compound, enough for triplicate wells each for:(1) VEGF (obtained from Pepro Tech Inc., catalogue no. 100-200, (2)endothelial cell growth factor (ECGF) (also known as acidic fibroblastgrowth factor, or aFGF) (obtained from Boehringer Mannheim Biochemica,catalogue no. 1439 600), or, (3) human PDGF B/B (1276-956, BoehringerMannheim, Germany) and assay media control. ECGF comes as a preparationwith sodium heparin.

[0680] 2. Transfer 50 μl/well of the test compound dilutions to the96-well assay plates containing the 0.8-1.0×10⁴ cells/100 μl/well of theHUV-EC-C cells from day 0 and incubate ˜2 h at 37° C., 5% CO₂.

[0681] 3. In triplicate, add 50 μl/well of 80 μg/ml VEGF, 20 ng/ml ECGF,or media control to each test compound condition. As with the testcompounds, the growth factor concentrations are 4× the desired finalconcentration. Use the assay media from day 0 step 2 to make theconcentrations of growth factors. Incubate approximately 24 hours at 37°C., 5% CO₂. Each well will have 50 μl test compound dilution, 50 μlgrowth factor or media, and 100 μl cells, which calculates to 200μl/well total. Thus the 4× concentrations of test compound and growthfactors become 1× once everything has been added to the wells.

[0682] DAY 2

[0683] 1. Add ³H-thymidine (Amersham, catalogue no. TRK-686) at 1μCi/well (10 μl/well of 100 μCi/ml solution made up in RPMI media+10%heat-inactivated fetal bovine serum) and incubate ˜24 h at 37° C., 5%CO₂. RPMI is obtained from Gibco BRL, catalogue no. 11875-051.

[0684] DAY 3

[0685] 1. Freeze plates overnight at −20° C.

[0686] DAY4

[0687] Thaw plates and harvest with a 96-well plate harvester (TomtecHarvester 96®) onto filter mats (Wallac, catalogue no. 1205-401), readcounts on a Wallac Betaplate™ liquid scintillation counter.

Vascular Permeability Assay

[0688] Increased vascular permeability in tumor-dependent angiogenesisis due to a loosening of gap junctions in response to vascularendothelial growth factor (VEGF). The Miles assay for vascularpermeability (Miles and Miles, J. Physiol. 118: 228-257 (1952)) has beenadapted to athymic mice in order to evaluate the ability of thecompounds of the present invention to inhibit VEGF-induced vascularpermeability in vivo. General Procedure:

[0689] Test compound or vehicle is administered prior to (typically itis 4 hours prior) to VEGF injection. 100μl of 0.5% Evan's blue dye inPBS is injected intravenously via lateral tail vein injections using a27 gauge needle. Sixty minutes later, animals are anesthetized using theinhalant Isofluorane. Following anesthesia, VEGF (100 ng of VEGF in 20μl of PBS) is injected intradermally in two spots and PBS (20 μl) isinjected in two spots in a grid pattern in the back of each animal. At adesignated timepoint of up to 1 hour after VEGF injection, the animalsare euthanized by CO₂ and the skin patches are dissected andphotographed. Based on a published report (Alicieri et al., Mol Cell 4:915-914 (1999)) quantitative evaluation of the VEGF-dependent dyeleakage into mouse skin can be achieved following elution of the dyefrom skin patches.

In Vivo Animal Models Xenograft Animal Models

[0690] The ability of human tumors to grow as xenografts in athymic mice(e.g., Balb/c, nu/nu) provides a useful in vivo model for studying thebiological response to therapies for human tumors. Since the firstsuccessful xenotransplantation of human tumors into athymic mice,(Rygaard and Povlsen, 1969, Acta Pathol. Microbial. Scand. 77:758-760),many different human tumor cell lines (e.g., mammary, lung,genitourinary, gastrointestinal, head and neck, glioblastoma, bone, andmalignant melanomas) have been transplanted and successfully grown innude mice. The following assays may be used to determine the level ofactivity, specificity and effect of the different compounds of thepresent invention. Three general types of assays are useful forevaluating compounds: cellular/catalytic, cellular/biological and invivo. The object of the cellular/catalytic assays is to determine theeffect of a compound on the ability of a TK to phosphorylate tyrosineson a known substrate in a cell. The object of the cellular/biologicalassays is to determine the effect of a compound on the biologicalresponse stimulated by a TK in a cell. The object of the in vivo assaysis to determine the effect of a compound in an animal model of aparticular disorder such as cancer.

[0691] Suitable cell lines for subcutaneous xenograft experimentsinclude C6 cells (glioma, ATCC # CCL 107), A375 cells (melanoma, ATCC #CRL 1619), A431 cells (epidermoid carcinoma, ATCC # CRL 1555), Calu 6cells (lung, ATCC # HTB 56), PC3 cells (prostate, ATCC # CRL 1435),SKOV3TP5 cells and NIH 3T3 fibroblasts genetically engineered tooverexpress EGFR, PDGFR, IGF-1R or any other test kinase. The followingprotocol can be used to perform xenograft experiments:

[0692] Female athymic mice (BALB/c, nu/nu) are obtained from SimonsenLaboratories (Gilroy, Calif.). All animals are maintained underclean-room conditions in Micro-isolator cages with Alpha-dri bedding.They receive sterile rodent chow and water ad libitum.

[0693] Cell lines are grown in appropriate medium (for example, MEM,DMEM, Ham's F10, or Ham's F12 plus 5%-10% fetal bovine serum (FBS) and 2mM glutamine (GLN)). All cell culture media, glutamine, and fetal bovineserum are purchased from Gibco Life Technologies (Grand Island, N.Y.)unless otherwise specified. All cells are grown in a humid atmosphere of90-95% air and 5-10% CO₂ at 37° C. All cell lines are routinelysubcultured twice a week and are negative for mycoplasma as determinedby the Mycotect method (Gibco).

[0694] Cells are harvested at or near confluency with 0.05% Trypsin-EDTAand pelleted at 450×g for 10 min. Pellets are resuspended in sterile PBSor media (without FBS) to a particular concentration and the cells areimplanted into the hindflank of the mice (8-10 mice per group, 2-10×10⁶cells/animal). Tumor growth is measured over 3 to 6 weeks using veniercalipers. Tumor volumes are calculated as a product of length × width ×height unless otherwise indicated. P values are calculated using theStudents t-test. Test compounds in 50-100 μL excipient (DMSO, orVPD:D5W) can be delivered by IP injection at different concentrationsgenerally starting at day one after implantation.

Tumor Invasion Model

[0695] The following tumor invasion model has been developed and may beused for the evaluation of therapeutic value and efficacy of thecompounds identified to selectively inhibit KDR/FLK-1 receptor.

[0696] Procedure

[0697] 8 week old nude mice (female) (Simonsen Inc.) are used asexperimental animals. Implantation of tumor cells can be performed in alaminar flow hood. For anesthesia, Xylazine/Ketamine Cocktail (100 mg/kgketamine and 5 mg/kg Xylazine) are administered intraperitoneally. Amidline incision is done to expose the abdominal cavity (approximately1.5 cm in length) to inject 10⁷ tumor cells in a volume of 100 μlmedium. The cells are injected either into the duodenal lobe of thepancreas or under the serosa of the colon. The peritoneum and musclesare closed with a 6-0 silk continuous suture and the skin is closed byusing wound clips. Animals are observed daily.

[0698] Analysis

[0699] After 2-6 weeks, depending on gross observations of the animals,the mice are sacrificed, and the local tumor metastases to variousorgans (lung, liver, brain, stomach, spleen, heart, muscle) are excisedand analyzed (measurement of tumor size, grade of invasion,immunochemistry, in situ hybridization determination, etc.).

Additional Assays

[0700] Additional assays which may be used to evaluate the compounds ofthis invention include, without limitation, a bio-flk-1 assay, an EGFreceptor-HER2 chimeric receptor assay in whole cells, a bio-src assay, abio-lck assay and an assay measuring the phosphorylation function ofraf. The protocols for each of these assays may be found in U.S.application Ser. No. 09/099,842, which is incorporated by reference,including any drawings, herein. Additionally, U.S. Pat. No. 5,792,783,filed Jun. 5, 1996 and U.S. application Ser. No. 09/322,297, filed May28, 1999 are incorporated by reference as if fully set forth herein.

Measurement of Cell Toxicity

[0701] Therapeutic compounds should be more potent in inhibitingreceptor tyro sine kinase activity than in exerting a cytotoxic effect.A measure of the effectiveness and cell toxicity of a compound can beobtained by determining the therapeutic index, i.e., IC₅₀/LD₅₀. IC₅₀,the dose required to achieve 50% inhibition, can be measured usingstandard techniques such as those described herein. LD₅₀, the dosagewhich results in 50% toxicity, can also be measured by standardtechniques as well (Mossman, 1983, J. Immunol. Methods, 65:55-63), bymeasuring the amount of LDH released (Korzeniewski and Callewaert, 1983,J. Immunol. Methods, 64:313, Decker and Lohmann-Matthes, 1988, J.Immunol. Methods, 115:61), or by measuring the lethal dose in animalmodels. Compounds with a large therapeutic index are preferred. Thetherapeutic index should be greater than 2, preferably at least 10, morepreferably at least 50.

Plasma Stability Test

[0702] The prodrug(3Z)-3-[(3,5-dimethyl-1H-pyrrol-2-yl)-methylidene]-1-(1-pyrrolidinylmethyl)-1,3-dihydro-2H-indol-2-onewas administered IV at 2 mg/mL to dogs. Levels of both prodrug and drug(3(Z)-3-[(3,5-dimethyl-1H-pyrrol-2-yl)-methylidene]-1,3-dihydro-2H-indol-2-one)were followed by HPLC analysis of blood plasma for 4 hours followingdosing. This study showed that the half-life for conversion of theprodrug to drug was 7.3 min. From a plot of drug concentration vs. time,the area under the curve indicated that 80% of the prodrug was convertedinto drug.

Formulation Examples

[0703] The formulations being Evaluated are Listed in Tables 1 and 2 andare Described Below

[0704] [A] Solid Formulations to be Reconstituted to a Stable Infusate(Table 1)

[0705] (1) Lyophilized Formulation

[0706] (a) Captisol Based: This formulation uses Captisol and an acidicagent to form an in situ salt at a pH of 1.5-2.0 to compound andlyophilize solutions of drug at concentrations of 20.0-25.0 mg/mL. Thelyophilized cake is reconstituted with an IV fluid to provide a stableinfusate at 2 mg/mL or higher at pH 3.

[0707] (b) Non-Captisol based: This formulation uses small amounts of asurfactant such as Polysorbate-80 or Cremophor EL and an acidic agent toform an in-situ salt at a pH of 1.5-2.0 to compound and lyophilizesolutions of drug at concentrations of 20.0-25.0 mg/mL. The lyophilizedcake is reconstituted with cosolvent-surfactant based aqueous diluentsuch as PEG-300-Polysorbate 80 or PEG-300-Cremophor EL to provide astable infusate at 2 mg/mL or higher at pH 3.

[0708] (2) Sterile API fill

[0709] The drug is filled as a sterile powder fill in a container andwill be reconstituted with a specific co-solvent—surfactant basedaqueous diluent to provide a stable infusate at 2 mg/mL or higher ofdrug at pH 3.

[0710] [B] Solution Concentrate to be Diluted to a Stable Infusate(Table 2)

[0711] The drug is solubilized in a non-aqueous mixture of co-solventsand surfactants at a high concentration such that it can be diluted withaqueous diluents to a stable infusate. The concentration of the drug inthe infusate is at a concentration of 2 mg/mL or higher, at pH 3. Thetotal levels of the co-solvent is less than 15% and the levels ofsurfactant is than 0.5%. TABLE 1 Formulation (1) Solid formulationsAttributes Lyophilized-Captisol based Lyophilized Non-Captisol basedSterile API Fill Dose/50 CC 200-300 200-300 300-400 vial (mgs) SterileAPI fill NA NA 300-400 mg in vial Composition- drug (mg) 200-300 drug(mg) 200-300 NA Lyophilized Acid (M) 1.4 Acid (M) 1.4 Cake Antioxidant(mg) 0-10 Antioxidant (mg) 0-10 Captisol (mg) 2000-3000 Filler (mg)200-300 Polysorbate-80 (mg) 0-50 Composition- 0.9% NaCl, D5W, PEG-300 (%w/v) 5-20 PEG-300 (% w/v) 5-20 Reconstitution Polysorbate-80 (% w/v)0-1.0 Polysorbate-80 (% w/v) 0-1.0 Fluid Citrate Buffer pH 3.0 CitrateBuffer pH 3.0 0.1M (% w/v) 30-40 0.1M (% w/v) 30-40 Water (qs to volume)Water (qs to volume) Composition- drug (mg/mL) 2-3 drug (mg/mL) 2-3 drug(mg/mL) 2-3 Reconstituted Acid (Molar) 1.4 Acid (Molar) 1.4 InfusateAcid (Molar) 1.4 Antioxidant (mgmL) 0-1.0 Antioxidant (mg/mL) 0-1.0(Administered Filler (mg/mL) 2-3 Filler (mg/mL) 2-3 to Patient)Antioxidant (mg/mL) 0-1.0 PEG-300 (mg/mL) 50-200 PEG-300 (mg/mL) 50-200Polysorbate-80 (mg/mL) 0-10 Polysorbate-80 (mg/mL) 0-10 Captisol (mg/mL)20-30 Citrate Buffer 0.3 Citrate Buffer 0.3 0.1M, pH 3.0 (mL) 0.1M, pH3.0 (mL) IV fluid (qs to volume) Water (qs to volume) Water (qs tovolume) pH 3.0 pH 3.0 pH 3.0 Composition- drug (mg/mL) 20-30 drug(mg/mL) 20-30 NA Prelyophilate Acid (Molar) 1.4 Acid (Molar) 1.4Antioxidant (mg/mL) 0-10 Filler (mg/mL) 20-30 Antioxidant (mg/mL) 0-10Polysorbate-80 (mg) 0-50 Water for Injection qs to 1.0 mL Captisol(mg/mL) 200-300 pH 1.5-2.0 Water for Injection qs to 1.0 mL pH 1.5-2.0

[0712] TABLE 2 Composition of Solution Formulation Composition (% w/v)or Ingredients mg/mL Drug 1.2-2.5 (12-25 mg/mL) Cosolvent - (PEG-300,PEG- 70-90 400) Surfactant 0-10 Dimethylacetamide 0-2 Salt forming agent(Methane Equivalent to 1.4M ratio of sulfonic acid, Tartaric acid, theAPI Citric Acid, Succinic Acid) Alcohol Qs to Volume

[0713] Formulation to be diluted 10 fold with pharmaceuticallyacceptable IV fluids.

[0714] The present invention is not to be limited in scope by theexemplified embodiments which are intended as illustrations of singleaspects of the invention, and any clones, DNA or amino acid sequenceswhich are functionally equivalent are within the scope of the invention.Indeed, various modifications of the invention in addition to thosedescribed herein will become apparent to those skilled in the art fromthe foregoing description and accompanying drawings. Such modificationsare intended to fall within the scope of the appended claims.

[0715] All references cited herein are hereby incorporated by referencein their entirety.

What is claimed is:
 1. A compound of the Formula (I):

wherein: R³, R⁴, R⁵ and R⁶ are independently selected from the groupconsisting of hydrogen, alkyl, trihaloalkyl, cycloalkyl, alkenyl,alkynyl, aryl, heteroaryl, heteroalicyclic, hydroxy, alkoxy, aryloxy,mercapto, alkylthio, arylthio, sulfinyl, sulfonyl, S-sulfonamido,N-sulfonamido, trihalomethane-sulfonamido, carbonyl, C-carboxy,O-carboxy, C-amido, N-amido, cyano, nitro, halo, O-carbamyl, N-carbamyl,O-thiocarbamyl, N-thiocarbamyl, amino and —NR¹¹R¹² where R¹¹ and R¹² areindependently selected from the group consisting of hydrogen, alkyl,cycloalkyl, aryl, carbonyl, acetyl, sulfonyl, andtrifluoromethanesulfonyl, or R¹¹ and R¹², together with the nitrogenatom to which they are attached, combine to form a five- or six-memberheteroalicyclic ring provided that at least two of R³, R⁴, R⁵ and R⁶ arehydrogen; or R³ and R⁴, R⁴ and R⁵, or R⁵ and R⁶ combine to form asix-member aryl ring, a methylenedioxy or an ethylenedioxy group; R⁷ isselected from the group consisting of hydrogen, alkyl, cycloalkyl,alkenyl, alkynyl, aryl, heteroaryl, heteroalicyclic, hydroxy, alkoxy,aryloxy, carbonyl, acetyl, C-amido, C-thioamido, amidino, C-carboxy,O-carboxy, sulfonyl, and trihalomethane-sulfonyl; R⁸, R⁹ and R¹⁰ areindependently selected from the group consisting of hydrogen, alkyl,trihaloalkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl,heteroalicyclic, hydroxy, alkoxy, aryloxy, mercapto, alkylthio,arylthio, sulfinyl, sulfonyl, S-sulfonamido, N-sulfonamido, carbonyl,C-carboxy, O-carboxy, cyano, nitro, halo, O-carbamyl, N-carbamyl,O-thiocarbamyl, N-thiocarbamyl, C-amido, N-amido, amino and —NR¹¹R¹²,wherein R¹¹ and R¹² are as defined above; or a pharmaceuticallyacceptable salt thereof.
 2. The compound of claim 1, wherein R⁷ ishydrogen.
 3. The compound of claim 1 wherein R³, R⁴, R⁵, R⁶, R⁷, and R⁹are hydrogen, R⁸ and R¹⁰ are unsubstituted lower alkyl.
 4. The compoundof claim 1, wherein R⁸ and R¹⁰ are methyl.
 5. The compound of claim 1,wherein the compound is


6. A pharmaceutical composition comprising a pharmaceutically acceptablecarrier or excipient and a compound of claim
 1. 7. A pharmaceuticalcomposition comprising a pharmaceutically acceptable carrier orexcipient and a compound of claim
 5. 8. The pharmaceutical compositionof claim 6, wherein said composition is administered parenterally. 9.The pharmaceutical composition of claim 5, wherein said composition isadministered parenterally.
 10. A method of treating a human having adisease capable of treatment by administration of a protein kinaseinhibitor, comprising administration to that human of a therapeuticallyeffective amount of a compound of claim
 1. 11. A method of treating ahuman having a disease capable of treatment by administration of aprotein kinase inhibitor, comprising administration to that human of atherapeutically effective amount of a compound of claim
 5. 12. Themethod of claim 11, wherein said disease is selected from the groupconsisting of cancer, blood vessel proliferative disorders, fibroticdisorders, mesangial cell proliferative disorders, metabolic diseasesand infectious diseases.
 13. The method of claim 12, wherein the canceris selected from the group consisting of colorectal cancer, Kaposi'ssarcoma and lung cancer.
 14. The method of claim 12, wherein the bloodvessel proliferative disorder is selected from the group consisting ofarthritis and restenosis.
 15. The method of claim 12, wherein thefibrotic disorder is selected from the group consisting of hepaticcirrhosis and atherosclerosis.
 16. The method of claim 12, wherein themesangial cell proliferative disorder is selected from the groupconsisting of glomerulonephritis, diabetic nephropathy, malignantnephrosclerosis, thrombotic microangiopathy syndromes, transplantrejection and glomerulopathies.
 17. The method of claim 12, wherein themetabolic disease is selected from the group consisting of psoriasis,diabetes mellitus, wound healing, inflammation and neurodegenerativediseases.
 18. A process of preparing a compound of Formula (I)comprising reacting a compound of Formula (II)

with formaldehyde and pyrrolidine.
 19. The process of claim 18, whereinR³-R⁷ and R⁹ are hydrogen and R⁸ and R¹⁰ are methyl.
 20. The process ofclaim 18, further comprising modifying any of the R³-R¹⁰ groups.
 21. Theprocess of claim 18, further comprising preparing an acid addition salt.